Methods for fast nucleic acid amplification

ABSTRACT

Methods, devices, and kits are provided for performing PCR in &lt;20 seconds per cycle, with improved efficiency and yield.

PRIORITY STATEMENT

This application is a continuation application of, and claims priorityto, U.S. application Ser. No. 16/179,383, filed Nov. 2, 2018, which is adivisional application of U.S. application Ser. No. 15/890,843, filedFeb. 7, 2018, now U.S. Pat. No. 10,144,960, issued Dec. 4, 2018, whichis a divisional application of U.S. application Ser. No. 14/403,369,filed Nov. 24, 2014, now U.S. Pat. No. 9,932,634, issued Apr. 3, 2018,which is a 35 U.S.C. § 371 national phase application of PCT ApplicationSerial No. PCT/US2013/042473, filed May 23, 2013, which claims thebenefit, under 35 U.S.C. § 119(e) of U.S. Provisional Patent ApplicationSer. No. 61/811,145, filed Apr. 12, 2013, and U.S. Provisional PatentApplication Ser. No. 61/651,161, filed May 24, 2012, the entire contentsof each of which are incorporated by referenced herein.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 1267-9TSDV2CT_ST25.txt, 7,435 bytes in size, generatedon Apr. 27, 2021 and filed via EFS-Web, is provided in lieu of a papercopy. This Sequence Listing is incorporated by reference into thespecification for its disclosures.

BACKGROUND OF THE INVENTION

Polymerase chain reaction (PCR) is a technique widely used in molecularbiology. It derives its name from one of its key components, a DNApolymerase used to amplify a piece of DNA by in vitro enzymaticreplication. As PCR progresses, the DNA generated (the amplicon) isitself used as a template for replication. This sets in motion a chainreaction in which the DNA template is exponentially amplified. With PCR,it is possible to amplify a single or few copies of a piece of DNAacross several orders of magnitude, generating millions or more copiesof the DNA piece. PCR employs a thermostable polymerase, dNTPs, and apair of primers.

PCR is conceptually divided into 3 reactions, each usually assumed tooccur over time at each of three temperatures. Such an “equilibriumparadigm” of PCR is easy to understand in terms of three reactions(denaturation, annealing, and extension) occurring at 3 temperaturesover 3 time periods each cycle. However, this equilibrium paradigm doesnot fit well with physical reality. Instantaneous temperature changes donot occur; it takes time to change the sample temperature. Furthermore,individual reaction rates vary with temperature, and once primerannealing occurs, polymerase extension immediately follows. Moreaccurate, particularly for rapid PCR, is a kinetic paradigm wherereaction rates and temperature are always changing. Holding thetemperature constant during PCR is not necessary as long as the productsdenature and the primers anneal. Under the kinetic paradigm of PCR,product denaturation, primer annealing, and polymerase extension maytemporally overlap and their rates continuously vary with temperature.Under the equilibrium paradigm, a cycle is defined by 3 temperatureseach held for a time period, whereas the kinetic paradigm requirestransition rates and target temperatures. Illustrative time/temperatureprofiles for the equilibrium and kinetic paradigms are shown in FIGS.15a-15b . However, it is understood that these temperature profiles areillustrative only and that in some implementations of PCR, the annealingand extension steps are combined so that only 2 temperatures are needed.

Paradigms are not right or wrong, but they vary in their usefulness. Theequilibrium paradigm is simple to understand and lends itself well tothe engineering mindset and instrument manufacture. The kinetic paradigmis more relevant to biochemistry, rapid cycle PCR, and melting curveanalysis.

When PCR was first popularized in the late 1980s, the process was slow.A typical protocol was 1 minute for denaturation at 94° C., 2 minutesfor annealing at 55° C., and 3 minutes for extension at 72° C. When thetime for transition between temperatures was included, 8 minute cycleswere typical, resulting in completion of 30 cycles in 4 hours.Twenty-five percent of the cycling time was spent in temperaturetransitions. As cycling speeds increased, the proportion of time spentin temperature transitions also increased and the kinetic paradigmbecame more and more relevant. During rapid cycle PCR, the temperatureis usually changing. For rapid cycle PCR of short products (<100 bps),100% of the time may be spent in temperature transition and no holdingtimes are necessary. For rapid cycle PCR of longer products, atemperature hold at an optimal extension temperature may be included.

In isolation, the term “rapid PCR” is both relative and vague. A 1 hourPCR is rapid compared to 4 hours, but slow compared to 15 minutes.Furthermore, PCR protocols can be made shorter if one starts with highertemplate concentrations or uses fewer cycles. A more specific measure isthe time required for each cycle. Thus, “rapid cycle PCR” (or “rapidcycling”) was defined in 1994 as 30 cycles completed in 10-30 minutes(1), resulting in cycles of 20-60 seconds each. This actual time of eachcycle is longer than the sum of the times often programmed fordenaturation, annealing and extension, as time is needed to ramp thetemperatures between each of these stages. Initial work in the early1990s established the feasibility of rapid cycling using capillary tubesand hot air for temperature control. Over the years, systems have becomefaster, and the kinetic requirements of denaturation, annealing, andextension have become clearer.

In one early rapid system, a heating element and fan from a hair dryer,a thermocouple, and PCR samples in capillary tubes were enclosed in achamber (2). The fan created a rapid flow of heated air past thethermocouple and capillaries. By matching the thermal response of thethermocouple to the sample, the temperature of the thermocouple closelytracked the temperature of the samples, even during temperature changes.Although air has a low thermal conductivity, rapidly moving air againstthe large surface area exposed by the capillaries was adequate to cyclethe sample between denaturation, annealing, and extension temperatures.Electronic controllers monitored the temperature, adjusted the power tothe heating element, and provided the required timing and number ofcycles. For cooling, the controller activated a solenoid that opened aportal to outside air, introducing cooling air to the otherwise closedchamber.

Temperatures could be rapidly changed using the capillary/air system.Using a low thermal mass chamber, circulating air, and samples in glasscapillaries, PCR products >500 bp were visualized on ethidium bromidestained gels after only 10 minutes of PCR (30 cycles of 20 seconds each)(3). Product yield was affected by the extension time and theconcentration of polymerase. With 30 second cycle times (about 10seconds between 70 and 80° C. for extension), the band intensityincreased as the polymerase concentration was increased from 0.1 to 0.8Units per 10 μl reaction. It is noted that polymerase Unit definitionscan be confusing. For native Taq polymerase, 0.4 U/10 μl is about 1.5 nMunder typical rapid cycling conditions (50).

Rapid protocols use momentary or “0” second holds at the denaturationand annealing temperatures. That is, the temperature-time profiles showtemperature spikes for denaturation and annealing, without holding thetop and bottom temperatures. Denaturation and annealing can occur veryquickly.

Rapid and accurate control of temperature allowed analytical study ofthe required temperatures and times for PCR. For an illustrative 536 bpfragment of human genomic DNA (β-globin), denaturation temperaturesbetween 91° C. and 97° C. were equally effective, as were denaturationtimes from <1 second to 16 seconds. However, it was found thatdenaturation times longer than 16 seconds actually decreased productyield. Specific products in good yield were obtained with annealingtemperatures of 50-60° C., as long as the time for primer annealing waslimited. That is, best specificity was obtained by rapid cooling fromdenaturation to annealing and an annealing time of <1 second. Yield wasbest at extension temperatures of 75-79° C., and increased withextension time up to about 40 seconds.

Conclusions from this early work were: 1) denaturation of PCR productsis very rapid with no need to hold the denaturation temperature, 2)annealing of primers can occur very quickly and annealing temperatureholds may not be necessary, and 3) the required extension time dependson PCR product length and polymerase concentration. Also, rapid cyclePCR is not only faster, but better in terms of specificity and yield (4,5) as long as the temperature was controlled precisely. PCR speed is notlimited by the available biochemistry, but by instrumentation that doesnot control the sample temperature closely or rapidly.

However, most current laboratory PCR instruments perform poorly withmomentary denaturation and annealing times, and many don't even allowprogramming of “0” second holding periods. Time delays from thermaltransfer through the walls of conical tubes, low surface area-to-volumeratios, and heating of large samples force most instruments to rely onextended times at denaturation and annealing to assure that the samplereaches the desired temperatures. With these time delays, the exacttemperature vs time course becomes indefinite. The result is limitedreproducibility within and high variability between commercial products(6). Many instruments show marked temperature variance duringtemperature transitions (7, 8). Undershoot and/or overshoot oftemperature is a chronic problem that is seldom solved by attemptedsoftware prediction that depends on sample volume. Such difficulties arecompounded by thermal properties of the instrument that may change withage.

Over time, conventional heat block instruments have become faster, withincremental improvements in “thin wall” tubes, more conductive heatdistribution between samples, low thermal mass blocks and other “fast”modifications. Nevertheless, it is unusual for these systems to cyclerapidly enough to complete a cycle in less than 60 seconds. A few heatblock systems can achieve <60 second cycles, usually restricted to2-temperature cycling between a limited range of temperatures. Byflattening the sample container, rapid cycling can be achieved byresistive heating and air cooling (9), or by moving the sample in aflexible tube between heating zones kept at a constant temperature (U.S.Pat. No. 6,706,617).

Commercial versions of the air/capillary system for PCR have beenavailable since 1991 (1) and for real-time PCR since 1996 (10, 11).Rapid cycling capabilities of other instruments are often comparedagainst the air/capillary standard that first demonstrated 20-60 secondcycles. Oddly enough, there has been a trend to run the capillary/airsystems slower over the years, perhaps reflecting discomfort with “0”second denaturation and annealing times by many users. Also,heat-activated enzymes require long activation periods, often doublingrun times even when “fast” activation enzymes are used. Anothercompromise away from rapid cycling is the use of plastic capillaries.These capillaries are not thermally matched to the instrument, so 20second holds at denaturation and annealing are often required to reachthe target temperatures (12).

Some progress in further decreasing the cycle times for PCR has occurredin microsystems, where small volumes are naturally processed (13, 14).However, even with high surface area-to-volume sample chambers, cyclesmay be long if the heating element has a high thermal mass and isexternal to the chamber (15). With thin film resistive heaters andtemperature sensors close to the samples, 10-30 minute amplification canbe achieved (16, 17).

While cooling of low thermal mass systems is usually by passive thermaldiffusion and/or by forced air, several interesting heating methods havebeen developed. Infrared radiation can be used for heating (18) withcalibrated infrared pyrometry for temperature monitoring (19).Alternatively, thin metal films on glass capillaries can serve as both aresistive heating element and a temperature sensor for rapid cycling(20). Finally, direct Joule heating and temperature monitoring of thePCR solution by electrolytic resistance is possible and has beenimplemented in capillaries (21). All of the above methods transfer heatto and from fixed samples.

Instead of heat transfer to and from stationary samples, the samples canbe physically moved to different temperature baths, or through channelswith fixed temperature zones. Microfluidic methods have become popular,with the PCR fluid passing within channels through different segmentskept at denaturation, annealing, and extension temperatures. Continuousflow PCR has been demonstrated within serpentine channels that pass backand forth through 3 temperature zones (22) and within loops ofincreasing or decreasing radius that pass through 3 temperature sectors(23). A variant with a serpentine layout uses stationary thermalgradients instead of isothermal zones, to more closely fit the kineticparadigm of PCR (24). To limit the length of the microchannel necessaryfor PCR, some systems shuttle samples back and forth between temperaturezones by bi-directional pressure-driven flow (25), pneumatics (26), orelectrokinetic forces (27). Instead of linear shuttling of samples, asingle circular channel can be used with sample movement driven as amagnetic ferrofluid (28) or by convection (29). One potential advantageof microsystem PCR, including continuous flow methods, is cycling speed.

Although some microsystems still require >60 second cycles, many operatein the 20-60 second cycle range of rapid cycle PCR (13, 30). Minimumcycle times ranging from 16-37 seconds have been reported for infraredheating (18, 19). Metal coated capillaries have achieved 40 second PCRcycles (20), while direct electrolytic heating has amplified with 21second cycles (20). Minimum cycle times reported for closed loopconvective PCR range from 24-42 seconds (29, 31). Several groups havefocused on reducing PCR cycle times to <20 seconds, faster than theoriginal definition of rapid cycle PCR that was first demonstrated in1990. Thin film resistive heating of stationary samples has reducedcycle times down to 17 seconds for 25 μl samples (32) and 8.5 secondsfor 100 μl samples (17). Continuous flow systems have achieved 12-14second cycles with thermal gradient PCR (24) and sample shuttling (26),while a ferrofluid loop claims successful PCR with 9 second cycles (28).Continuous flow systems through glass and plastic substrates haveachieved cycle times of 6.9 seconds (22) and 5.2 seconds (23) forvarious size PCR products. Alternating hot and cool water conductionthrough an aluminum substrate amplified 1 μl droplets under oil with5.25 second cycles (33). Similarly, water conduction through a porouscopper block amplified 5 μl samples with 4.6 second cycles (34). Acontinuous flow device of 1 μl reaction plugs augmented by vaporpressure achieved 3 second cycles (35). Additionally, there are reportsthat claim to amplify an 85 bp fragment of the Stx bacteriophage of E.coli in capillaries with 2.7 second cycles by immersion of thecapillaries in gallium sandwiched between Peltier elements (36).Alternatively, PCR amplification in capillaries cycled by pressurizedhot and cool gases obtained 2.6 second cycles (48).

Table 1 summarizes work to minimize PCR cycle times to less than the 20second cycles that originally defined “Rapid PCR”. Over the past 20years, new prototype instruments have been developed that incrementallyimprove cycling speed. However, practical PCR performance (efficiencyand yield) is often poor. As a general rule, as cycles becomeincreasingly shorter, claims for successful PCR correlate with lowercomplexity targets (bacteria, phage, multicopy plasmids, or even PCRproducts) that are used at higher starting concentrations (see, e.g.,U.S. Pat. No. 6,210,882, wherein 5 ng of amplicon was used as thestarting sample). Indeed, none of the studies listed in Table 1 with <20second cycles used complex eukaryotic DNA such as human DNA. Thestarting copy number of template molecules is often very high (e.g.,180,000,000 copies of lambda phage/μl), so that little amplification isneeded before success is claimed. Furthermore, the lack of no templatecontrols in many studies raises questions regarding the validity ofpositive results, especially in an environment with high templateconcentrations. One instrument-oriented report focuses extensively onthe design and modeling of the thermal cycling device, with a finalbrief PCR demonstration using a high concentration of a low complexitytarget. Heating and cooling rates (up to 175° C./s) have been reportedbased on modeling and measurements without PCR samples present (17).

TABLE 1 Fastest Cycle Total Product Time [Template] Template [Primers][Polymerase] Length (s) (Copies/μl) Form (nM) Polymerase (nM) (bp) 201,600 Human 1000 0.08 3 536 DNA U/μl Taq 12 40,000 Lambda 400 0.2 7.5500 phage U/μl Taq 12 1,000,000 230 bp PCR 1000 0.5 19 230 product U/μlTaq 9.25 4,700- 18S rDNA 1800 Taq Gold ? 187 470,000 (human genomic) 918,000,000 Lambda 2000 0.025 0.94 500 phage U/μl Taq 8.5 ?  cDNA 1800 ?? 82 7.0 10,000,000 1 KB PCR 2000 0.25 9.4 176 product U/μl Taq 6.310,000 Plasmids 1200 0.05 U/μl ? 134 (B. anthracis) Ex Taq HS 5.2/9.7180,000,000 Lambda 400 0.07 2.6 500/997 phage U/μl Taq 5.25 1,400,000 B.subtilis 500 0.025 U/μl ? 72 (bacterial KOD plus DNA) 4.6 34,000 E.herbicola 800 0.04 U/μl 4  58/160 (bacterial KAPA2G DNA) 4.2 50¹ B.subtilis ?  KOD plus ? 72 (bacterial 0.05 DNA) U/μl Ex 3.0 10,000Plasmids 1200 Taq HS ? 134 (B. anthracis) 2.7 ?  stx phage ?³ KOD ? 85(E. coli) 2.6 ?⁴ stx phage ?⁵ 0.5 19 85 (E. coli) U/μl Taq Fastest CycleNo Time Template (s) Quantification Trend Method Control? Reference 20Faint Gel Increases with Capillary Air No 3 Band [Polymerase] CyclingCapillary 12 Electro- ? IR Heating, No 56 phoresis Pressurized AirCooling 12 Good gel Dependent Continuous Yes 55 band on cycle # Flow andcopy # 9.25 ? ? IR Heating of No 54 droplets in oil 9 OK gel bandIntensity Continuous No 28 increases with Flow with a cycle time FerrousParticle Plug 8.5 80% Decreasing Micromachined ?  17 efficiencyefficiency cantilever at faster cycles 7.0 7% of 50% at 15 s ContinuousYes 22 control cycles Flow 6.3 55% of ? Plug Yes 53 control ContinuousFlow 5.2/9.7 Faint gel Dependent on Continuous No 23 bands cycle timesFlow 5.25 90% Single run Water pumped Yes 33 efficiency against (SYBR)aluminum plate 4.6 Faint gel Yield increases Water pumped No 31 bandswith # cycles through porous copper 4.2 Cq = 33 Higher copy # IR laser?² 51 (SYBR) reduces Cq 3.0 15% of 80% at 7.5 s Constant flow Yes (5% 35control cycles with vapor signal) pressure 2.7 Barely Decreasing Galliumtransfer No 36 visible yield from from Peltiers band 3.06 s to tocapillaries 2.69 s cycles 2.6 Very dim Constant from Pressurized gas No48 band 2.8 to and capillaries 2.6 s cycles ¹Presumed single copy in a20 nl droplet with Cq of 33 under SYBR Green monitoring, but no gel ormelting analysis to confirm PCR product identity. ²A “Blank” sample wasrun, but it is not clear if this was a no template control. ³Articlesays [primer] is 0.5 mmol, patent application (US 2009/0275014 A1) says[primer] is 0.01-0.5 μM. ⁴Two pg E. coil DNA/μl, but copy number ofphage in the DNA preparation is unknown. ⁵Dissertation says 0.5 μmol/10μl (50 mM), patent (U.S. Pat. No. 6,472,186) says 50 pmol/10 μl (5 μM).

One way to decrease cycle time is to introduce variations to the PCRprotocol to ease the temperature cycling requirements. Longer primerswith higher Tms allow higher annealing temperatures. By limiting theproduct length and its Tm, denaturation temperatures can be lowered tojust above the product Tm. In combination, higher annealing and lowerdenaturation temperatures decrease the temperature range required forsuccessful amplification. Reducing 3-step cycling (denaturation,annealing, and extension) to 2-steps (denaturation and a combinedannealing/extension step) also simplifies the temperature cyclingrequirements. Both decreased temperature range and 2-step cycling aretypical for the studies in Table 1 with cycle times <20 seconds.Two-step cycling can, however, compromise polymerase extension rates ifthe combined annealing/extension step is performed at temperatures lowerthan the 70 to 80° C. temperature optimum where the polymerase is mostactive. Polymerase extension rates are log-linear with temperature untilabout 70-80° C., with a reported maximum of 60-120 bp/s (50).

Even with protocol variations, amplification efficiency and yield areoften poor when cycle times are <20 seconds when compared to controlreactions (22, 23). These efforts towards faster PCR appear dominated byengineering with little focus on the biochemistry. As cycle timesdecrease from 20 seconds towards 2 seconds, PCR yield decreases andfinally disappears, reflecting a lack of robustness even with simpletargets at high copy number.

The instrumentation in various references disclosed in Table 1 may besuitable for extremely fast PCR, if reaction conditions are compatible.As disclosed herein, a focus on increased concentrations of primers,polymerase, and Mg++ allows for “extreme PCR” (PCR with <20 secondcycles (30 cycles in <10 min)), while retaining reaction robustness andyield.

SUMMARY OF THE INVENTION

In one aspect of the present invention a method for amplifying a targetnucleic acid sequence in a biological sample during amplification isprovided, the method comprising the steps of adding a thermostablepolymerase and primers configured for amplification of the targetnucleic acid sequence to the biological sample, wherein the polymeraseis provided at a concentration of at least 0.5 μM and primers are eachprovided at a concentration of at least 2 μM, and amplifying the targetnucleic acid sequence by polymerase chain reaction by thermally cyclingthe biological sample between at least a denaturation temperature and anelongation temperature through a plurality of amplification cycles usingan extreme temperature cycling profile wherein each cycle is completedin less than 20 seconds per cycle.

In another aspect of the invention, a method for amplifying a targetnucleic acid sequence in a biological sample during amplification isprovided, the method comprising the steps of adding a thermostablepolymerase and primers configured for amplification of the targetnucleic acid sequence to the biological sample, wherein the polymeraseto primer ratio is (about 0.03 to about 0.4 polymerase):(total primerconcentration), and the polymerase concentration is at least 0.5 μM; andamplifying the target nucleic acid sequence by polymerase chain reactionby thermally cycling the biological sample between at least adenaturation temperature and an elongation temperature through aplurality of amplification cycles using an extreme temperature cyclingprofile wherein each cycle is completed in less than 20 seconds.

In yet another aspect of this invention, a device for performing PCR isprovided, comprising a sample container for holding a PCR sample, meansfor heating the sample, means for cooling the sample, and control meansfor repeatedly subjecting the sample container to the means for heatingthe sample and the means for cooling the sample to thermal cycle thesample at a ramp rate of at least 200° C./s.

In still one more embodiment, kits for performing PCR on a targetnucleic acid are provided, the kits comprising: dNTPs, a polymeraseprovided at a concentration of at least 0.5 μM, and a pair of primersconfigured for amplifying the target nucleic acid, wherein the primersare each provided at a concentration of at least 2 μM.

Additional features of the present invention will become apparent tothose skilled in the art upon consideration of the following detaileddescription of preferred embodiments exemplifying the best mode ofcarrying out the invention as presently perceived.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1a shows a schematic for performing extreme PCR.

FIG. 1b is an illustrative device for performing extreme PCR withreal-time capabilities for monitoring one sample tube in a water bath.

FIG. 1c is an illustrative device for performing extreme PCR withthree-temperature cycling.

FIG. 1d is a close up view of the optics of the device in FIG. 1b thatalso shows the temperature reference capillary.

FIG. 2a is a graph that superimposes the location of the sample holder(-----) of FIG. 1b with the temperature of the sample (—).

FIG. 2b is a temperature graph of extreme PCR using the device shown inFIG. 1 b.

FIG. 2c is a temperature graph of rapid cycle PCR using a carouselLightCycler shown for comparison against FIG. 2 b.

FIG. 3a shows derivative melting curves of extreme PCR products (-----)and rapid cycle PCR products (—Ψ⋅—), with negative controls for extreme(—) and rapid (—-—) cycling, amplified using the temperature profile ofFIG. 2 b.

FIG. 3b is a 2% SeaKem LE agarose gel of the same samples of FIG. 3a ,lanes 1 and 8 are size markers, lanes 2 and 3 are products resultingfrom 30 sec extreme PCR, lane 4 is a no template control for 30 secextreme PCR, lanes 5 and 6 are products resulting from 12 min PCR, andlane 7 is the no template control for 12 min PCR.

FIG. 3c shows an extreme PCR temperature trace (-----) that amplifiedthe same products shown in FIGS. 3a and 3b , along with real-timemonitoring (—) of the same reaction.

FIG. 4a shows an extreme PCR temperature trace that increases theextension rate by temperature control.

FIG. 4b shows a magnified portion of FIG. 4a , superimposing thelocation of the sample holder (—) of FIG. 1b with the temperature of thesample (-----).

FIG. 4c is a negative derivative melting curve (−dF/dT) of a 58 bpamplicon of IRL10RB, wherein AA (—), AG (—-—), and GG (-----) genotypesare shown.

FIG. 5a is a three dimensional graph plotting polymerase concentrationvs. primer concentration vs. concentration of PCR product, using extremePCR.

FIG. 5b is the extreme PCR temperature trace used in FIG. 5 a.

FIG. 5c shows negative derivative melting curves of the 4 μM Klentaqpolymerase (KT POL) products from FIG. 5 a.

FIG. 5d is an agarose gel showing results of extreme PCR using varyingpolymerase concentrations at 10 μM primer concentrations from FIG. 5 a.

FIG. 6a is a temperature trace of extreme PCR performed in a 19 gaugestainless steel tube.

FIG. 6b is a gel of the PCR products produced by the extreme temperaturecycles of FIG. 6 a.

FIG. 7a is an extreme PCR temperature trace with a long (1 second)combined annealing/extension step.

FIG. 7b is a three dimensional graph plotting polymerase concentrationvs. primer concentration vs. concentration of PCR product, using extremePCR for a 102 bp product.

FIG. 8a shows an extreme PCR temperature profile used to amplify a 226bp product, using a one second combined annealing/extension step.

FIG. 8b shows an extreme PCR temperature profile used to amplify a 428bp product, using a four second combined annealing/extension step.

FIG. 8c shows the real time results obtained from FIG. 8a and a similartemperature trace using a 2 second annealing/extension step, includingno template controls for each.

FIG. 8d shows the real time results obtained from FIG. 8b and a similartemperature trace using a 5 second annealing/extension step, includingno template controls for each.

FIG. 9a shows amplification curves of a 45 bp fragment of KCNE1 atdifferent starting concentrations.

FIG. 9b is a plot of Cq versus log₁₀ (initial template copies) of thedata from FIG. 9a . Reactions were performed in quintuplicate.

FIGS. 9c-9d are similar to FIGS. 9a -9b, except showing amplification ofa 102 bp fragment of NQO1.

FIG. 10a is a three dimensional graph plotting polymerase concentrationvs. primer concentration vs. concentration of PCR product, using extremePCR for a 300 bp product (20 cycles, 4.9 seconds per cycle).

FIG. 10b shows fluorescence versus cycle number plots for PCRamplification of a 500 bp synthetic template using KAPA2G FASTpolymerase and 1-5 second extension times.

FIG. 10c is a plot of extension length vs. minimum extension time forseveral KlenTaq polymerase concentrations and KAPA2G FAST polymerase.

FIGS. 11a-11e show fluorescence versus cycle number plots for PCRamplification of products of size: 100 bp (FIG. 11a ), 200 bp (FIG. 11b), 300 bp (FIG. 11c ), 400 bp (FIGS. 11d ), and 500 bp (FIG. 11e ).

FIG. 12a shows negative derivative melting curves of a 60 bp fragment ofAKAP10 after 35 cycles of extreme PCR, using varying magnesiumconcentrations.

FIG. 12b is a gel of the PCR products shown in the negative derivativemelting curves of FIG. 12 a.

FIG. 13a shows negative derivative melting curves of a 60 bp fragment ofAKAP10 after 35 cycles, using varying cycle times with 5 mM Mg⁺⁺. Cycletimes were 0.32 seconds (—), 0.42 seconds (—⋅⋅—), 0.52 seconds (—-—),and 0.62 seconds (-----). Cycle times included a 0.1 to 0.4 second holdin a 60° bath.

FIG. 13b is a gel of the PCR products shown in the negative derivativemelting curves of FIG. 13 a.

FIG. 14a shows negative derivative melting curves of a 60 bp fragment ofAKAP10, as amplified on three different instruments: (1) extreme PCR,(2) LightCycler (Roche), and (3) CFX96 (BioRad).

FIG. 14b is a gel of the PCR products shown in the negative derivativemelting curves of FIG. 14 a.

FIGS. 15a -15b show illustrative profiles for an equilibrium paradigm(FIG. 15a ) and a kinetic paradigm (FIG. 15b ) of PCR. Solid blackrepresents denaturation, striped represents annealing, and solid whiterepresents extension of the nucleic acids during thermal cycling.

DETAILED DESCRIPTION

As used herein, the terms “a,” “an,” and “the” are defined to mean oneor more and include the plural unless the context is inappropriate.Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. The term “about” is usedherein to mean approximately, in the region of, roughly, or around. Whenthe term “about” is used in conjunction with a numerical range, itmodifies that range by extending the boundaries above and below thenumerical values set forth. In general, the term “about” is used hereinto modify a numerical value above and below the stated value by avariance of 5%. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by use ofthe antecedent “about,” it will be understood that the particular valueforms another embodiment. It will be further understood that theendpoints of each of the ranges are significant both in relation to theother endpoint, and independently of the other endpoint.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

By “sample” is meant an animal; a tissue or organ from an animal; a cell(either within a subject, taken directly from a subject, or a cellmaintained in culture or from a cultured cell line); a cell lysate (orlysate fraction) or cell extract; a solution containing one or moremolecules derived from a cell, cellular material, or viral material(e.g. a polypeptide or nucleic acid); or a solution containing anaturally or non-naturally occurring nucleic acid, which is assayed asdescribed herein. A sample may also be any body fluid or excretion (forexample, but not limited to, blood, urine, stool, saliva, tears, bile)that contains cells, cell components, or nucleic acids.

The phrase “nucleic acid” as used herein refers to a naturally occurringor synthetic oligonucleotide or polynucleotide, whether DNA or RNA orDNA-RNA hybrid, single-stranded or double-stranded, sense or antisense,which is capable of hybridization to a complementary nucleic acid byWatson-Crick base-pairing. Nucleic acids of the invention can alsoinclude nucleotide analogs (e.g., BrdU, dUTP, 7-deaza-dGTP), andnon-phosphodiester internucleoside linkages (e.g., peptide nucleic acid(PNA) or thiodiester linkages). In particular, nucleic acids caninclude, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or anycombination thereof.

By “probe,” “primer,” or “oligonucleotide” is meant a single-strandedDNA or RNA molecule of defined sequence that can base-pair to a secondDNA or RNA molecule that contains a complementary sequence (the“target”). The stability of the resulting hybrid depends upon thelength, GC content, nearest neighbor stacking energy, and the extent ofthe base-pairing that occurs. The extent of base-pairing is affected byparameters such as the degree of complementarity between the probe andtarget molecules and the degree of stringency of the hybridizationconditions. The degree of hybridization stringency is affected byparameters such as temperature, salt concentration, and theconcentration of organic molecules such as formamide, and is determinedby methods known to one skilled in the art. Probes, primers, andoligonucleotides may be detectably-labeled, either radioactively,fluorescently, or non-radioactively, by methods well-known to thoseskilled in the art. dsDNA binding dyes (dyes that fluoresce morestrongly when bound to double-stranded DNA than when bound tosingle-stranded DNA or free in solution) may be used to detect dsDNA. Itis understood that a “primer” is specifically configured to be extendedby a polymerase, whereas a “probe” or “oligonucleotide” may or may notbe so configured.

By “specifically hybridizes” is meant that a probe, primer, oroligonucleotide recognizes and physically interacts (that is,base-pairs) with a substantially complementary nucleic acid (forexample, a sample nucleic acid) under high stringency conditions, anddoes not substantially base pair with other nucleic acids.

By “high stringency conditions” is meant conditions that allowhybridization comparable with that resulting from the use of a DNA probeof at least 40 nucleotides in length, in a buffer containing 0.5 MNaHPO4, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at atemperature of 65° C., or a buffer containing 48% formamide, 4.8×SSC,0.2 M Tris-Cl, pH 7.6, 1×Denhardt's solution, 10% dextran sulfate, and0.1% SDS, at a temperature of 42° C. Other conditions for highstringency hybridization, such as for PCR, northern, Southern, or insitu hybridization, DNA sequencing, etc., are well known by thoseskilled in the art of molecular biology (47).

In an illustrative embodiment, methods and kits are provided for PCRwith <20 second cycle times, with some embodiments using <10 second, <5second, <2 second, <1 second, and <0.5 second cycle times. With thesecycle times, a 30 cycle PCR is completed in <10 min, <5 min, <2.5 min,<1 min, <30 seconds, and <15 seconds, respectively. As PCR speeds becomeincreasingly faster, the primer and polymerase concentrations areincreased, thereby retaining PCR efficiency and yield.

Compromising any of the 3 component reactions of PCR (primer annealing,polymerase extension, and template denaturation) can limit theefficiency and yield of PCR. For example, if primers anneal to only 95%of the template, the PCR efficiency cannot be greater than 95%, even if100% of the templates are denatured and 100% of the primed templates areextended to full length products. Similarly, if extension is only 95%efficient, the maximum possible PCR efficiency is only 95%. In order forthe PCR product concentration to double each cycle, all the componentsmust reach 100% completion. Denaturation, annealing and extension willbe considered sequentially in the following paragraphs.

Inadequate denaturation is a common reason for PCR failure, in slow (>60second cycles), rapid (20-60 second cycles), and extreme (<20 secondcycles) PCR temperature cycling. The goal is complete denaturation eachcycle, providing quantitative template availability for primerannealing. Initial denaturation of template before PCR, particularlygenomic DNA, usually requires more severe conditions than denaturationof the amplification product during PCR. The original optimization ofrapid cycle PCR (4) was performed after boiling the template, a good wayto assure initial denaturation of genomic DNA. Incomplete initialdenaturation can occur with high Tm targets, particularly those withflanking regions of high stability (37). This can compromisequantitative PCR, illustratively for genomic insertions or deletions,particularly if minor temperature differences during denaturation affectPCR efficiency (37-39). If prior boiling or restriction digestion (37)is not desired, and higher denaturation temperatures compromise thepolymerase, adjuvants that lower product Tm can be used to help withdenaturation.

Although 94° C. is often used as a default target temperature fordenaturation, it is seldom optimal. PCR products melt over a 40° C.range, depending primarily on GC content and length (43). Lowdenaturation target temperatures have both a speed and specificityadvantage when the PCR product melts low enough that a lowerdenaturation temperature can be used. The lower the denaturationtemperature, the faster the sample can reach the denaturationtemperature, and the faster PCR can be performed. Added specificityarises from eliminating all potential products with higher denaturationtemperatures, as these potential products will remain double-strandedand will not be available for primer annealing. To amplify high Tmproducts, the target temperature may need to be increased above 94° C.However, most current heat stable polymerases start to denature above97° C. and the PCR solution may boil between 95 and 100° C., dependingon the altitude, so there is not much room to increase the temperature.Lowering the monovalent salt and Mg′ concentration lowers product Tm.Similarly, incorporating dUTP and/or 7-deaza-dGTP also lowers productTm, but may decrease polymerase extension rates. Most proprietary PCR“enhancers” are simple organics that lower product Tm, enablingdenaturation (and amplification) of high Tm products. Most popular amongthese are DMSO, betaine, glycerol, ethylene glycol, and formamide. Inaddition to lowering Tm, some of these additives also raise the boilingpoint of the PCR mixture (particularly useful at high altitudes). As theconcentration of enhancer increases, product Tms decrease, butpolymerase inhibition may increase.

Denaturation, however, need not be rate limiting even under extremecycling conditions, because DNA unwinding is first order and very fast(10-100 msec), even when the temperature is only slightly above theproduct Tm. Denaturation occurs so rapidly at 2-3° C. above the Tm ofthe amplification product that it is difficult to measure, but completedenaturation of the amplicon probably occurs in less than 0.1 second. Ifthe product melts in multiple domains, the target denaturationtemperature should be 2-3° C. above the highest melting domain. As longas the sample reaches this temperature, denaturation is very fast, evenfor long products. Using capillaries and water baths (40), completedenaturation of PCR products over 20 kB occurred in less than one second(52). Product Tms and melting domains are illustratively determinedexperimentally with DNA dyes and high resolution melting (41). AlthoughTm estimates can be obtained by software predictions (42), theiraccuracy is limited. Furthermore, observed Tms strongly depend on localreaction conditions, such as salt concentrations and the presence of anydyes and adjuvants. Thus, observed Tms are usually better matched to thereaction conditions.

Without any effect on efficiency, the approach rate to denaturation canbe as fast as possible, for example 200-400° C./s, as shown in FIGS. 2aand 6a . At these rates, only about 0.1-0.2 seconds are required toreach denaturation temperatures. However, a slower rate as the targettemperature is approached decreases the risk of surpassing the targettemperature and avoids possible polymerase inactivation or boiling ofthe solution. One illustrative method to achieve a slower approachtemperature is to submerge the sample in a hot bath that exceeds thetarget temperature by 5-10° C. The temperature difference between thetarget and bath temperatures determines the exponential approach curvethat automatically slows as the difference decreases. By continuouslymonitoring the temperature, the next phase (cooling toward annealing) istriggered when the denaturation target is achieved. In summary, completeproduct denaturation in PCR requires <0.2 s at temperatures 2-3° C.above the highest melting domain temperature of the product and thedenaturation temperature can be approached as rapidly as possible,illustratively at 40-400° C./second. Since denaturation is first order,its rate depends only on the product concentration, and the efficiency(or percentage of the product that is denatured) is independent of theproduct concentration.

Incomplete and/or misdirected primer annealing can result in poor PCR.Low efficiency results if not all template sites are primed.Furthermore, if priming occurs at undesired sites, alternative productsmay be produced. The goal is essentially complete primer annealing toonly the desired sites each cycle, providing quantitative primedtemplate for polymerase extension.

Rapid PCR protocols with 20-60 second cycles suggest an annealing timeof <1 second at 5° C. below the Tm of 500 nM primers (52). Primerconcentrations for instruments attempting <20 second cycles range from200-1,000 nM each (Table 1). These concentrations are similar to thoseused in conventional PCR (>60 second cycles), where long annealing timesare used. Lowering the primer concentration is often used to improvespecificity, and increasing the primer concentration is seldomconsidered due to concerns regarding nonspecific amplification. However,with rapid cycling, improved specificity has been attributed to shorterannealing times (5). If this trend is continued, one would expect thatvery short annealing times of extreme PCR should tolerate high primerconcentrations. To promote annealing, an annealing temperature 5° C.below the primer Tm is recommended for 20-60 second cycles. Tms are bestmeasured experimentally by melting analysis using saturating DNA dyesand oligonucleotides under the same buffer conditions used foramplification. The primer is combined with its complementary target witha 5′-extension as a dangling end, to best approximate the stability of aprimer annealed to its template, and melting analysis is performed.

In contrast to denaturation, annealing efficiency depends on the primerconcentration. Primer annealing can become limiting at very fast cyclespeeds. Primer annealing is a second order reaction dependent on bothprimer and target concentrations. However, during most of PCR, theprimer concentration is much higher than the target concentration andannealing is effectively pseudo-first order and dependent only on theprimer concentration. In this case, the fraction of product that isprimed (the annealing efficiency) depends only on the primerconcentration, not the product concentration, so that higher primerconcentrations should allow for shorter annealing times. Furthermore,without being bound to theory, it is believed that the relationship islinear. As the annealing time becomes shorter and shorter, increasedprimer concentrations become necessary to maintain the efficiency andyield of PCR. For example, rapid cycling allows about 1-3 seconds forannealing at temperatures 5° C. below primer Tm (3). If this annealingtime (at or below Tm−5° C.) is reduced 10-fold in extreme PCR, a similarpriming efficiency would be expected if the primer concentration wereincreased 10-fold. As the available annealing time becomes increasinglyshorter, the primer concentration should be made increasingly higher byapproximately the same multiple. Typical rapid PCR protocols use 500 nMeach primer. If the annealing time in extreme PCR is reduced 3 to40-fold, the primer concentrations required to obtain the same primingefficiency are 1,500-20,000 nM each primer. This is equivalent to3,000-40,000 nM total primers, higher than any primer concentration inTable 1. This suggests that one reason for poor efficiency in priorattempts at <20 second cycling is poor annealing efficiency secondary toinadequate primer concentrations. In extreme PCR, the primerconcentrations are increased to 1.5-20 μM each to obtain excellentannealing efficiency despite annealing times of 0.05-0.3 seconds. Evergreater primer concentrations can be contemplated for ever shorterannealing times, using increased primer concentrations to offsetdecreased annealing times to obtain the same annealing efficiency. It isnoted that most commercial instruments require a hold time of at least 1second, while a few instruments allow a hold time of “0” seconds, but nocommercial instrument allows a hold time of a fractional second. Forsome illustrative examples of extreme PCR, hold times in increments of0.1 or 0.01 seconds may be desirable.

Another way to increase the annealing rate and shorten annealing timeswithout compromising efficiency is to increase the Mg⁺⁺ concentration.Annealing rates are known in the art to increase with increasing ionicstrength, and divalent cations are particularly effective for increasingrates of hybridization, including primer annealing.

Illustratively, the approach rate to the annealing target temperaturemay be as fast as possible. For example, at 200-800° C./s (FIGS. 2a and6a ), annealing temperatures can be reached in 0.05-0.2 seconds. Rapidcooling also minimizes full length product rehybridization. To theextent that duplex amplification product forms during cooling, PCRefficiency is reduced because primers cannot anneal to the duplexproduct. Although this is rare early in PCR, as the productconcentration increases, more and more duplex forms during cooling.Continuous monitoring with SYBR® Green I suggests that such productreannealing can be a major cause of the PCR plateau (44).

Polymerase extension also requires time and can limit PCR efficiencywhen extension times are short. Longer products are known to requirelonger extension times during PCR and a final extension of severalminutes is often appended at the end of PCR, presumably to completeextension of all products. The usual approach for long products is tolengthen the time for extension. Using lower extension temperaturesfurther increases required times, as in some cases of 2-step cyclingwhere primer annealing and polymerase extension are performed at thesame temperature.

Essentially complete extension of the primed template each cycle isrequired for optimal PCR efficiency. Most polymerase extension ratesincrease with temperature, up to a certain maximum. For Taq polymerase,the maximum is about 100 nucleotides/s at 75-80° C. and it decreasesabout 4-fold for each 10° C. that the temperature is reduced (50). For a536 bp beta-globin product, 76° C. was found optimal in rapid cycle PCR(4). Faster polymerases have recently been introduced with commercialclaims that they can reduce overall PCR times, suggesting that they maybe able to eliminate or shorten extension holding times for longerproducts.

As an alternative or complement to faster polymerase extension rates, ithas been found that increasing the concentration of polymerase reducesthe required extension time. Given a standard Taq polymeraseconcentration in PCR (0.04 U/μl) or 1.5 nM (49) with 500 nM of eachprimer, if each primer is attached to a template, there is only enoughpolymerase to extend 0.15% of the templates at a time, requiringrecycling of the polymerase over and over again to new primed templatesin order to extend them all. By increasing the concentration ofpolymerase, more of the available primed templates are extendedsimultaneously, decreasing the time required to extend all thetemplates, presumably not by faster extension rates, but by extending agreater proportion of the primed templates at any given time.

To a first approximation, for small PCR products (<100 bp), the requiredpolymerization time appears to be directly proportional to thepolymerization rate of the enzyme (itself a function of temperature) andthe polymerase concentration. The required time is also inverselyproportional to the length of template to be extended (product lengthminus the primer length). By increasing the polymerase activity 20-300fold over the standard activity of 0.04 U/μl in the PCR, extreme PCRwith <20 second cycles can result in high yields of specific products.That is, activities of 0.8-12 U/μl (1-16 μM of KlenTaq) enable two-stepextreme PCR with combined annealing/extension times of 0.1-1.0 second.The highest polymerase activity used previously was 0.5 U/μl (Table 1).For two-step PCR that is used in illustrative examples of extreme PCR, acombined annealing/extension step at 70-75° C. is advantageous forfaster polymerization rates. Furthermore, because it simplifiestemperature cycling, two-step PCR is typically used in illustrativeexamples of extreme cycling (<20 second cycles) and both rapid annealingand rapid extension must occur during the combined annealing/extensionstep. Therefore, both increased primer concentrations and increasedpolymerase concentrations are used in illustrative examples, resultingin robust PCR under extreme two-temperature cycling. Illustratively,primer concentrations of 1.5-20 μM each and polymerase concentrations of0.4-12 U/μl of any standard polymerase (0.5-16 μM of KlenTaq) arenecessary with combined annealing/extension times of 0.05-5.0 seconds at50-75° C., as illustrated in the Examples to follow. Because there isonly one PCR cycling segment for both annealing and extension, extremePCR conditions require enhancement of both processes, illustratively byincreasing the concentrations of both the primers and the polymerase.

Extreme three-temperature cycling is also envisioned, where theannealing and extension steps are kept separate at differenttemperatures. In this case, the time allotted to annealing and extensionsteps can be individually controlled and tailored to specific needs. Forexample, if only the annealing time is short (0.05-0.2 seconds) and theextension time is kept comparatively long (illustratively for 1, 2, 5,10 or 15 seconds), only the primer concentrations need to be increasedfor efficient PCR. Alternatively, if the extension time is short (<1 secwithin 70-80° C.), but the annealing time is long, it is believed thatonly the polymerase concentration needs to be increased to obtainefficient PCR. It is understood that efficient PCR has an illustrativeefficiency of at least 70%, more illustratively of at least 80%, andeven at least 90%.

For products longer than 100 bp, efficient extension using extreme PCRmay need a combination of high polymerase concentration and increasedextension time. If the polymerase is in excess, the minimum timeillustratively should be the extension length (defined as the productlength minus the primer length) in bases divided by the polymeraseextension rate in bases/second. However, as previously noted, thepolymerase is usually only saturating in the beginning of PCR, beforethe concentration of template increases to greater than theconcentration of polymerase. One way to decrease cycle time is to usetwo-temperature PCR near the temperature maximum of the polymerase,typically 70-80° C. The required extension time can be determinedexperimentally using real-time PCR and monitoring the quantificationcycle or Cq. For example, at a polymerase extension rate of 100bases/second at 75° C., a 200 bp product would be expected to requireabout 2 seconds if the concentration of polymerase is in excess.Similarly, a 400 bp product would be expected to require about 4 secondsusing this same polymerase as long as its concentration is greater thanthe template being extended. If the polymerase is not in excess, addingmore polymerase allows more templates to be extended at the same time,decreasing the required extension time in proportion to theconcentration of polymerase.

The utility of any DNA analysis method depends on how fast it can beperformed, how much information is obtained, and how difficult it is todo. Compared to conventional cloning techniques, PCR is fast and simple.Rapid cycle and extreme PCR focus on continued reduction of the timerequired. Real-time PCR increases the information content by acquiringdata each cycle. Melting analysis can be performed during or after PCRto monitor DNA hybridization continuously as the temperature isincreased.

Returning to the equilibrium and kinetic paradigms of PCR (FIG. 15a-15b), extreme PCR of products <100 bps exemplifies a good applicationof the kinetic model. Temperatures are always changing and rates ofdenaturation, annealing, and extension depend on temperature, so anadequate assessment of PCR can only be obtained by integrating the ratesof the component reactions across temperature. For products greater than100 bp, longer extension times may be necessary, and components of boththe kinetic and equilibrium models are appropriate.

When the reaction conditions are configured according to at least oneembodiment herein, it has been found that PCR can be performed at veryfast rates, illustratively with some embodiments in less than one minutefor complete amplification, with cycle times of less than two seconds.Illustratively, various combinations of increased polymerase and primerconcentrations are used for this extreme PCR. Without being bound to anyparticular theory, it is believed that an excess concentration ofprimers will allow for generally complete primer annealing, therebyincreasing PCR efficiency. Also without being bound to any particulartheory, it is believed that an increase in polymerase concentrationimproves PCR efficiency by allowing more complete extension. Increasedpolymerase concentration favors binding to the annealed primer, and alsofavors rebinding if a polymerase falls off prior to complete extension.The examples below show that extreme PCR has been successful, even whenstarting with complex eukaryotic genomic DNA.

Although KlenTaq was used in the Examples to follow, it is believed thatany thermostable polymerase of similar activity will perform in asimilar manner in extreme PCR, with allowances for polymerase extensionrates. For example, Herculase, Kapa2G FAST, KOD Phusion, natural orcloned Thermus aquaticus polymerase, Platinum Taq, GoTaq and Fast Startare commercial preparation of polymerases that should enable extreme PCRwhen used at the increased concentrations presented here, illustrativelyadjusted for differences in enzyme activity rates.

Because no current commercial PCR instrument allows for two second cycletimes, a system 4 was set up to test proof of concept for extreme PCR.However, it is understood that the system 4 is illustrative and othersystems that can thermocycle rapidly are within the scope of thisdisclosure. As shown in FIG. 1a , a hot water bath 10 of 95.5° C. (thetemperature of boiling water in Salt Lake City, Utah, the location wherethe present examples were performed), and a cool water bath 14 of 30-60°C. are used to change the temperature of 1-5 μl samples contained in asample container 20. The illustrative water baths 10, 14 are 4.5 quartstainless steel dressing jars (Lab Safety Supply, #41634), although 500ml glass beakers were used in some examples, and are heated on electrichotplates 12, 16 with magnetic stirring (Fisher Scientific IsotempDigital Hotplates (#11-300-49SHP). However, it is understood that otherembodiments may be used to heat and cool the samples. In the embodimentshown in FIG. 1a , the sample container 20 is a composite glass/plasticreaction tube (BioFire Diagnostics #1720, 0.8 mm ID and 1.0 mm OD).However, in other examples, hypodermic needles (Becton Dickenson#305187, 0.042″ ID, 0.075″ OD) and composite stainless steel/plasticreaction tubes constructed from stainless steel tubing (Small Parts,0.042″ ID/0.075″ OD, 0.035″ ID/0.042″ OD, or 0.0265″ ID/0.035″ OD) andfit into the plastic tops of the BioFire tubes were used as the samplecontainer 20. While other sample containers are within the scope of thisinvention, it is desirable that the sample containers have a largesurface area to volume ratio and have a fast heat transfer rate. Forcertain embodiments, the open end of the metal tubing was sealed byheating to a red-white color using a gas flame and compressing in avise. For real-time PCR, tubes that are optically clear or have anoptically clear portion are desirable. Samples were spun down to thebottom of each tube by brief centrifugation.

The sample container 20 is held by a tube holder 22 attached to astepper motor shaft 26 by arm 21. The tube holder 22 was machined fromblack Delrin plastic to hold 2-5 sample containers 20 (only one samplecontainer 20 is visible in FIG. 1a , but a row of such sample containers20 may be present) so that the reaction solutions were held at a radiusof 6.5-7.5 cm. While not visible in FIG. 1a , a thermocouple (Omega typeT precision fine wire thermocouple #5SRTC-TT-T-40-36, 36″ lead, 0.003′diameter with Teflon insulation) may be used to measure temperature.With reference to FIG. 1d , which shows a similar tube holder and arm ofFIG. 1b with like numbers representing similar components, a tube holder222 designed to hold two sample containers is present, with one locationin tube holder 222 occupied by a thermocouple 228. It is understood thatany number of sample containers 20 or 220 may be used in any of theembodiments described herein, with or without a thermocouple, as shownin FIG. 1d . Thermocouple amplification and linearization is performedwith an Analog Devices AD595 chip (not shown). The thermocouple voltagewas first calculated from the AD595 output as Type T voltage =(AD595output/247.3)−11 μV. Then the thermocouple voltage was converted totemperature using National Institute of Standards and Technologycoefficients for the voltage/temperature correlation of Type Tthermocouples. The analog signal was digitized (PCIe-6363 acquisitionboard) and processed by LabView software (version 2010, NationalInstruments) installed on CPU 40 and viewed on user interface 42.Stepper motion illustratively is triggered dynamically at 87-92° C. and60-75° C. or may be held in each water bath for a computer-controlledperiod of time. Thirty to fifty cycles are typically performed.

The stepper motor 24 (Applied Motion Products, #HT23-401, 3V,3A) ispositioned between the water baths 10 and 14 so that all samplecontainers 20 in the tube holder 22 could flip between each water bath10 and 14, so that the portion of each sample container 20 containingsamples are completely submerged. The stepper motor 24 is poweredillustratively by a 4SX-411 nuDrive (National Instruments, not shown)and controlled with a PCI-7344 motion controller and NI-Motion Software(version 8.2, National Instruments) installed on CPU 40. Stepper motor24 rotates between water baths 10 and 14 in about 0.1 second. FIG. 2ashows a sample temperature trace (—) juxtaposed over a trace of theposition of the sample container 20 (-----) for a run where steppermotion was triggered at 90° C. and 50° C. As can be seen in FIG. 2,there is some overshoot to a temperature lower than 50° C., presumablydue do the time required to move the sample container 20 out of waterbath 14. Thus, as discussed above, it may be desirable to triggerstepper motor 24 at a somewhat higher temperature. In the examplesbelow, the temperatures given are for the sample temperature reached,not the trigger temperature. The maximum heating rate calculated fromFIG. 2a is 385° C./s and maximum cooling rate 333° C./s. Illustratively,extreme PCR may be performed with ramp rates of at least 200° C./s. Inother embodiments, the ramp rate may be 300° C./s or greater.

In some examples, system 4 is also configured for real-time monitoring.As shown in FIG. 1a , for real time monitoring, a fiber optics tip 50 ofoptics block 25 is mounted above sample container 20, such that whensample container 20 is being moved from hot water bath 10 to the coldwater bath by stepper motor 24, sample container 20 passes by the fiberoptics tip 50, with or without a hold in this monitoring position. Inthis illustrative embodiment, fiber optics tip is provided in air abovethe water baths. Thermocycling device 4 may be controlled by CPU 40 andviewed on user interface 42

FIG. 1b shows an embodiment similar to FIG. 1a . Hot plates 212 and 216are provided for controlling temperature of hot water bath 210 and coldwater bath 214. A stepper motor 224 is provided for moving samplecontainer 220 and thermocouple 228 (shown in FIG. 1d ), by moving arm221 and tube holder 222, which is illustratively made of aluminum.However, in this embodiment, the tip 250 of the fiber optics cable 252is held in water bath 214 by positioning block 254. Fiber optics cable252 enters water bath 214 through port 248 and provides signal to opticsblock 225. Thermocycling device 204 may be controlled by CPU 240 andviewed on user interface 242

Light from an Ocean Optics LLS-455 LED Light Source 256 was guided byfiber optics cable 252 (Ocean Optics P600-2-UV-VIS, 600 μm fiber corediameter) into a Hamamatsu Optics Block 258 with a 440+/−20 nmexcitation interference filter, a beamsplitting 458 nm dichroic and a490+/−5 nm emission filter (all from Semrock, not shown). Epifluorescentillumination of the capillary was achieved with another fiber opticcable (not shown) placed approximately 1-2 mm distant from and in-linewith the one sample capillary when positioned in the cooler water bath.Emission detection was with a Hamamatsu PMT 62.

FIG. 1c shows an illustrative system 304 for three-temperature PCR. Ahot water bath 310 of 95.5° C., a cool water bath 314 of 30-60° C., anda medium water bath 313 of 70-80° C. are used to change the temperatureof 1-5 μl samples contained in a sample container 320, and are heated onthree electric hotplates 312, 316, and 318 with magnetic stirring. Thesample container 320 is held by a tube holder 322 attached to a steppermotor 324 by arm 321. Thermocouple 328 is also held by tube holder 322.Arm 321 may be raised as stepper motor 324 rotates. A fiber optics tip350 is illustratively provided in medium water bath 313, although it isunderstood that it may be placed in air, as with FIG. 1a . Due to theset-up of this illustrative embodiment, it was not possible to place thethree water baths, 310, 313, and 314 equidistant from one another.Accordingly, the largest space was placed between hot water bath 310 andcool water bath 314, as cooling of the sample between these baths isdesirable, whereas the sample moves between the other water baths to beheated. However, it is understood that this configuration isillustrative only and that other configurations are within the spirit ofthis disclosure. Because two stepper motors are used simultaneously (oneto raise the capillary out of the water and one to transfer betweenwater baths) the angular motion of each can be minimized to decrease thetime of movement between baths. In the 2 water bath system, the requiredangular motion of the stepper to transfer the sample between baths isgreater than 270 degrees. However, in the 3 water bath system, thestepper motor that raises the samples needs to traverse less than 45degrees while the stepper moving the samples between water baths needsto move only 90 degrees or less. The water baths can also be configuredas sectors of a circle (pie-shaped wedges) to further limit the angularmovement required. Minimizing the angular movement decreases thetransfer time between water baths. Transfer times less than 100 msec oreven less than 50 msec are envisioned. Other components of this system304 are similar to the systems 4, 204 shown in FIGS. 1a-b and are notshown in FIG. 1 c.

Example 1

Unless otherwise indicated, PCR was performed in 5 μl reaction volumescontaining 50 mM Tris (pH 8.3, at 25° C.), 3 mM MgCl₂, 200 μM each dNTP(dATP, dCTP, dGTP, dTTP), 500 μg/ml non-acetylated bovine serum albumin(Sigma), 2% (v/v) glycerol (Sigma), 50 ng of purified human genomic DNA,and 1×LCGreen® Plus (BioFire Diagnostics). The concentration of theprimers and the polymerase varied according to the specific experimentalprotocols. Klentaq™ DNA polymerase was obtained from either AB Peptides,St. Louis, Mo., or from Wayne Barnes at Washington University (St.Louis). The molecular weight of KlenTaq is 62.1 kD with an extinctioncoefficient at 280 nm of 69,130 M⁻¹ cm⁻¹, as calculated from thesequence (U.S. Pat. No. 5,436,149). Mass spectrometry confirmed apredominate molecular weight of 62 kD, and denaturing polyacrylamidegels showed that the major band was greater than 80% pure byintegration. Using the absorbance and purity to calculate theconcentration indicated an 80 μM stock in 10% glycerol. Final polymeraseconcentrations were typically 0.25-16 μM. One μM KlenTaq is theequivalent of 0.75 U/μl, with a unit defined as 10 nmol of productsynthesized in 30 min at 72° C. with activated salmon sperm DNA. Primerswere synthesized by the University of Utah core facility, desalted, andconcentrations determined by A₂₆₀. The final concentrations of eachprimer typically varied from 2.5-20 μM.

A 45 bp fragment of KCNE1 was amplified from human genomic DNA usingprimers CCCATTCAACGTCTACATCGAGTC (SEQ ID NO:1) and TCCTTCTCTTGCCAGGCAT(SEQ ID NO:2). The primers bracketed the variant rs#1805128 (c.253G>A)and amplified the sequence:CCCATTCAACGTCTACATCGAGTCC(G/A)ATGCCTGGCAAGAGAAGGA (SEQ ID NO:3).

FIG. 3a shows a melting curve of the PCR product generated by extremePCR using the device shown in FIG. 1a , where 0.64 μM KlenTaq and 10 μMof each primer were used, and cycled between 91° C. and 50° C., as shownin FIG. 2b , for 35 cycles and a total amplification time of 28 seconds.Each cycle required 0.8 seconds. Also shown in FIG. 3a is a meltingcurve of the same amplicon generated by rapid cycling in theLightCycler, where 0.064 μM KlenTaq and 0.5 μM of each primer were used,and cycling was between 90° C. and 50° C. for 35 cycles and a totalamplification time of 12 minutes (FIG. 2c ). Each cycle required 10.3seconds. Note that because of the different time scales in FIGS. 2b and2c , the entire extreme PCR protocol of FIG. 2b is completed in lessthan 2 cycles of its rapid cycle counterpart. Both reactions producedamplicons having similar Tms and strong bands on gel electrophoresis(FIG. 3b ), whereas neither negative control showed amplification byeither melting analysis or gel electrophoresis. In this illustrativeexample, extreme PCR conditions showed greater yield than rapid cyclePCR conditions when analyzed on gels (FIG. 3b ). The 0.5° C. differencein Tm on the melting curves is believed to be due to the differentamounts of glycerol in each reaction, arising from the glycerol contentin the polymerase storage buffer (final concentration of glycerol in thePCR was 1.3% under extreme conditions and 0.1% under rapid conditions).FIG. 3b also confirms that the size of the amplicons were similar and aspredicted. In addition, despite the high concentrations of polymeraseand primers, the reaction appears specific with no indication ofnonspecific products. However, high resolution melting analysis wasunable to distinguish the 3 genotypes. The stoichiometric percentage ofpolymerase to total primer concentration was 3% for extreme PCR and 6.4%for rapid cycle PCR.

Real-time monitoring of the 45 bp KCNE1 reaction was performed using 1μM polymerase, 10 μM of each primer, and 1.3% glycerol. The sample wasmonitored each cycle in air between the 2 water baths using the deviceof FIG. 1a . The enclosed chamber air temperature was held at 70° C. andthe sample was interrogated for 0.2 seconds each cycle. As measured bythe temperature reference capillary, samples were cycled between 60 and90° C., as shown in FIG. 3c . The cycle time increased from 0.8 secondsto 1.12 seconds because of the added time for positioning and measuring.Thus, fifty cycles were completed in 56 seconds. Amplification wasapparent from an increase in fluorescence at about 30 cycles or afterabout 34 seconds (FIG. 3c ). The temperature remained near 60° C. whilethe sample was in air for measurement, limiting the extension rate ofthe polymerase.

As seen in FIG. 3c , this reaction has a quantification cycle (Cq) ofabout 25 cycles, but it does not seem to plateau until at least 50cycles. Also, because the reaction was stopped after 64 cycles, it ispossible that the quantity of amplicon may continue to increase and notplateau until significantly later. Without being bound to theory, it isbelieved that the increase in primer concentration allows for improvedyield and delayed plateau, illustratively 20 cycles after Cq, and moreillustratively 25 cycles or more after Cq.

Example 2

In this example, a 58 bp fragment bracketing an A>G variant (rs#2834167)in the interleukin 10 beta receptor was amplified with primersCTACAGTGGGAGTCACCTGC (SEQ ID NO:4) and GGTACTGAGCTGTGAAAGTCAGGTT (SEQ IDNO:5) to generate the following amplicon:CTACAGTGGGAGTCACCTGCTTTTGCC(A/G)AAGGGAACCTGACTTTCACAGC TCAGTACC (SEQ IDNO:6). Extreme PCR was performed as described in Example 1 using theinstrument shown in FIG. 1a . One μM polymerase, 10 μM each primer and1.3% glycerol were used (polymerase to total primer percentage=5%). Inorder to increase the temperature for polymerase extension to 70-80° C.,where the polymerase has higher extension rates, a different positioningprotocol was used. After reaching the annealing temperature, instead ofimmediately positioning in air for monitoring, the sample wastransferred to the hot water bath until the extension temperature wasreached. Then the sample was positioned in air just above the hot waterbath, producing the temperature cycles shown in FIGS. 4a and 4b , andenabling faster polymerase extension at optimal temperatures between 70and 77° C. The 3 different genotypes were each amplified by extreme PCRusing 0.97 second cycles, completing 39 cycles in 38 seconds. Afterextreme PCR, high resolution melting curves were obtained for eachgenotype on an HR-1 instrument modified to accept LC24 capillaries. FIG.4c reveals that all three genotypes were amplified and distinguished, asexpected.

Example 3

The reaction mixtures in Example 1 were the same for both the extremePCR and rapid cycle PCR, except for the amounts of polymerase andprimers, and a minor difference in glycerol concentration thatapparently caused the shift in Tm seen in FIG. 3a . In this and allfuture examples, the glycerol concentration was held at 2% by equalizingits concentration as necessary. For extreme PCR, 1 μM polymerase and 10μM of each primer were used, while for rapid cycle PCR, 0.064 μMpolymerase and 0.5 μM of each primer were used. As discussed above, itis believed that faster annealing times provide for improved primerspecificity. With this improved specificity, increased concentrations ofprimers may be used, which is believed to favor primer binding and allowreduced annealing times. Similarly, increased polymerase concentrationsfavor binding to the annealed primer, and also favor rebinding to theincomplete amplicon if a polymerase falls off prior to completeextension. In addition, because of the higher polymerase concentration,a greater proportion of the primed templates can be extended at onceeven late in PCR, reducing the number of templates that a singlepolymerase must extend and reducing the overall extension time.

FIG. 5a summarizes the results of extreme PCR cycling with variouspolymerase and primer concentrations. In this example, a 49 bp fragmentof the interleukin 10 beta receptor was amplified with primersGGGAGTCACCTGCTTTTGCC (SEQ ID NO:7) and TACTGAGCTGTGAAAGTCAGGTTCC (SEQ IDNO:8) and 3 mM MgCl₂, to generate:GGGAGTCACCTGCTTTTGCCAAAGGGAACCTGACTTTCACAGCTCAGTA (SEQ ID NO:9). Foreach extreme PCR reaction, the device shown in FIGS. 1b was used withoutreal time monitoring. The temperature was cycled between 90° C. and 63°C. for 35 cycles, for a total reaction time of just under 26 seconds(0.73 second cycles) as shown in FIG. 5b . Reaction conditions were asdiscussed in Example 1, except that the amounts of polymerase andprimers were varied, as shown in FIG. 5a . The vertical axis in FIG. 5ais quantified as the peak of the negative derivative plot of the meltingcurve, obtained without normalization on the HR-1 instrument. At 0.5 μMpolymerase, virtually no amplification was seen at any level of primerconcentration. However, at 1.0 μM polymerase, discernible levels ofamplification were seen at primer concentrations of 5 μM and above. Asthe polymerase levels increase, so do the amount of amplicon, up tolevels of about 4 μM. At 8 μM polymerase, the amount of ampliconplateaued or dropped off, depending on the primer concentration, with asignificant drop off at 16 μM at lower primer concentrations. It appearsthat under these extreme temperature cycling conditions for a 49 bpproduct, the polymerase has a favored concentration range between about1 and 8 μM, and more specifically between 2 and 8 μM, depending on theprimer concentration.

Similarly, little amplification was seen with primer concentrations of2.5 μM. However, amplification was successful at 5 μM primer, withKlenTaq concentrations of 2-8 μM, and amplification continued to improvewith increasing concentrations. Excellent amplification was achievedwith primer concentrations of about 10-20 μM primer. FIG. 5c showsmelting curves for various primer concentrations at 4 μM KlenTaq, whileFIG. 5d verifies the size of the product as the polymerase concentrationvaries while the primer concentration is held at 10 μM. Despite the highconcentrations of polymerase and primers, no nonspecific amplificationis seen.

Without being bound to theory, it appears that the ratio between theamount of enzyme and amount of primer is important for extreme PCRcycling, provided that both are above a threshold amount. It is notedthat the above amounts are provided based on each primer. Given that thepolymerase binds to each of the duplexed primers, the total primerconcentration may be the most important. For KlenTaq, suitable ratiosare 0.03-0.4 (3-40% enzyme to total primer concentration), with anillustrative minimum KlenTaq concentration of about 0.5 μM, and moreillustratively about 1.0 μM, for extreme PCR. The primers may beprovided in equimolar amounts, or one may be provided in excess, as forasymmetric PCR. The optimal polymerase:primer percentage may also dependon the temperature cycling conditions and the product size. For example,standard (slow) temperature cycling often uses a much lower polymeraseto primer percentage, typically 1.5 nM (0.04 U/μl) polymerase (49) and1,000 nM total primer concentration, for a percentage of 0.15%, over 10times lower than the percentages found effective for extreme PCR.

Example 4

The same PCR target as in Example 3 was amplified with 8 μM polymeraseand 20 μM each primer in a 19 gauge steel hypodermic needle, to increasethermal transfer and cycling speeds. The polymerase to total primerpercentage was 20%. Amplification was performed on the instrument ofFIG. 1b and was completed in 16 seconds using 35 cycles of 0.46 secondseach (FIG. 6a ), cycling between 91° C. and 59-63° C. The maximumheating rate during cycling was 407° C./s and the maximum cooling ratewas 815° C./s, demonstrating that PCR can occur with ramp rates ofgreater than 400° C./s with no holds. Analysis of the products on a 4%NuSieve 3:1 agarose gel revealed strong specific bands of the correctsize (FIG. 6b ). The no template control showed no product at 49 bp, butdid show a prominent primer band similar to the positive samples.

Example 5

A 102 bp fragment of the NQO1 gene was amplified using primersCTCTGTGCTTTCTGTATCCTCAGAGTGGCATTCT (SEQ ID NO:10) andCGTCTGCTGGAGTGTGCCCAATGCTATA (SEQ ID NO:11) and the instrument of FIG.1b without the real-time components. The polymerase concentration wasvaried between 0.25 and 4 μM, while each primer concentration was variedbetween 0.5 and 8 μM. The primers were designed to anneal at highertemperatures (low 70s) so that extension at a combinedannealing/extension phase would be at a more optimal temperature for thepolymerase. Greater polymerization rates at these temperatures wereexpected to enable amplification of longer products. The cooler waterbath was controlled at 72° C. and the end of the annealing/extensionphase triggered by time (1 second), rather than temperature. Cyclingbetween 72 and 90° C. for 30 cycles required 58 seconds using 1.93second cycles (FIG. 7a ). As seen in FIG. 7a , the sample temperaturedrops about 3° C. below the annealing/extension temperature while ittravels through the air to the hot water bath. FIG. 7b shows the amountof product amplified by quantifying the melting curves as in FIG. 5a .Melting curve analysis showed only a single product of Tm 84° C. Verylittle product was observed at 0.25 μM polymerase or at 1 μM eachprimer. Some amplification occurs at 2 μM each primer, with the bestamplification at 2-4 μM polymerase and 8 μM each primer. At primerconcentrations of 2-4 μM, yield decreases as the polymeraseconcentration increases, although this was not seen at 8 μM primerconcentration. Although the thermal cycling and target length aredifferent from Example 3, the best amplification occurs at polymerase tototal primer concentrations of 3.1 to 50%.

Example 6

Extreme PCR was used to amplify 135 bp and 337 bp fragments of the BBS2gene using the instrument shown in FIG. 1b with real time monitoring. Inorder to study the effect of product length on extreme PCR and controlfor possible confounding effects of different primers, the fragmentswere first amplified from genomic DNA using primers with common 5′-endextensions. For the 135 bp fragment the primers wereACACACACACACACACACACACACACACACACACACAAAAATTCAGTGGCAT TAAATACG (SEQ IDNO:12) and GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAAAACCAGAGCTAAAGGGAAG (SEQ ID NO:13). For the 337 bp fragment the primerswere ACACACACACACACACACACACACACACACACACACAAAAAGCTGGTGTCTG CTATAGAACTGATT(SEQ ID NO:14) and GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAAAAGTTGCCAGAGCTAAAGGGAAGG (SEQ ID NO:15). After standard PCR amplificationfrom genomic DNA, primers and dNTPs were degraded by ExoSAP-IT(Affymetrix, CA), followed by PCR product purification using theQuickStep™ 2 PCR Purification Kit (Catalog #33617, Edge BioSystems,Gaithersburg, Md.). PCR products were diluted approximately 1million-fold and adjusted to equal concentrations by equalizing the Cqobtained by standard real-time PCR to obtain a Cq of 25 cycles(approximately 10,000 copies/10 μl reaction).

Extreme PCR was performed on 1,000 copies of the amplified templates ina total volume of 5 μl using the common primersACACACACACACACACACACACACACACACACACACAAAAA (SEQ ID NO:16) andGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAAAA (SEQ ID NO:17) eachat 2 μM with 2 μM polymerase and 2% glycerol. The 135 bp BBS2 fragmentresulted in a 226 bp product requiring extension of 176 or 185 bases(depending on the primer), while the 337 bp BBS2 fragment resulted in a428 bp PCR product requiring extension of 378 or 387 bases. Specificamplification was verified on agarose gels and by melting analysis. Theextreme PCR temperature profile used for the 226 bp product is shown inFIG. 8a , which included a 1 second combined annealing/extension at 75°C. and denaturation at 87° C. Also performed was a 2 secondannealing/extension phase at the same temperature (trace not shown).Real time PCR results for these amplifications are shown in FIG. 8c ,revealing about a 5 cycle shift to higher Cq with the 1 second extensionas compared to the 2 second extension, presumably reflecting a decreasein efficiency as the extension time is decreased. The extreme PCRtemperature profile used for the 428 bp product is shown in FIG. 8b ,showing a 4 second combined annealing/extension at 75° C. anddenaturation at 87° C. Also performed was a 5 second annealing/extensionphase at the same temperature (trace not shown). Real time PCR resultsfor these amplifications are shown in FIG. 8d , revealing about a 2cycle shift to higher Cq with the 4 second extension as compared to the5 second extension, presumably reflecting a decrease in efficiency asthe extension time is decreased.

Example 7

Quantitative performance of PCR was assessed using the real-timeinstrument of FIG. 1b for the 102 bp fragment of NQO1 of Example 5 andthe 45 bp fragment of KCNE1 of Example 1 using a dilution series ofhuman genomic DNA, using 2 μM KlenTaq and 8 μM each primer for NQO1 and8 μM KlenTaq and 20 μM each primer for KNCE1. With a dynamic range of atleast 4 decades, as seen in FIGS. 9a and 9b , the amplificationefficiencies calculated from the standard curves were 95.8% for NQO1 and91.7% for KCNE1. Control reactions without template did not amplifyafter 50 cycles and single copy replicates (mean copy number of 1.5copies per reaction) were similar in amplification curve shape andintensity to higher concentrations (FIGS. 9A and 9C). At a mean copynumber of 0.15 copies/reaction, 2 reactions were positive out of 17(combining both NQO1 and KCNE1 trials), with a calculated expectation of0.13 copies/reaction by binomial expansion.

Example 8

The extension time required for different product lengths usingreal-time PCR (FIG. 10a-c ). To control for the possible confoundingeffects of different primers, synthetic templates of 100-500 bp usingthe following common high Tm (77° C.) primers:

(SEQ ID NO: 18) ACTCGCACGAACTCACCGCACTCC and (SEQ ID NO: 19)GCTCTCACTCGCACTCTCACGCACA.The synthetic template sequences were:

100 bp Template: (SEQ ID NO: 20)ACTCGCACGAACTCACCGCACTCCGGATGGATTGTGAAGAGGCCCAAGATACTGGTCATATTATCCTTTGATCTAGCTCTCACTCGCACTCTCACGCA CA. 200 bp Template:(SEQ ID NO: 21) ACTCGCACGAACTCACCGCACTCCTCAATGCTGACAAATCGAAAGAATAGGAATAGCGTAATTACTAGAGGACTCCAATATAGTATATTACCCTGGTGACCGCCTGTACTGTAGGAACACTACCGCGGTTATATTGACAGCTTAGCAATCTACCCTGTTGGGATCTGTTTAAGTGGCTCTCACTCGCACTCTCACG CACA. 300 bp Template:(SEQ ID NO: 22) ACTCGCACGAACTCACCGCACTCCCCTTCGAATATAAAGTACGACATTACTAGCAATGACAGTTCCAGGATTTAAGAAAGTAGTGTTCCACATCAATGCATATCCAGTGAAAGCATAACGTCAAAAAAAGCCTGGCACCGTTCGCGATCTGGACTTACTTAGATTTGTTGTAGTCAAGCCGGCTATCAGCGATTTATCCCGGAAACACATACTAGTGAGTTATTTGTATGTTACCTAGAATAGCTGTCACGAATCACTAATACATTCACCCACCAGCTCTCACTCGCACTCTCA CGCACA.400 bp Template: (SEQ ID NO: 23)ACTCGCACGAACTCACCGCACTCCTGAATACAAGACGACAGTCCTGATTATATTTTCATTTAATTACGCCAATTTAATTATGATGAATATTAACGGAATTAAATATGTATTGATAAGTACTAAGTAATGGTTTACCCACGGCGATCTATATGCAAGGGAAACATTAACAAATTTAAACATCTGATGTGGACAAAACTTGTAATGTGGTATAGTTAAAAATATAGGTTTCAGGGACACGTAAGTATCTATCTTGAATGTTTAAGTAGGTCCTGTCTACCATTCTGAAATTTAGAAAATCGCGTTCATCGGGCTGTCGGCTACACCTCAGAAAACCATTTCGTGTTGCACAGGAGGAACTTTCGAGGGTTCGTATGAGCTCTCACTCGCACTCT CACGCACA.500 bp Template: (SEQ ID NO: 24)ACTCGCACGAACTCACCGCACTCCACCGCTTGACGACGTAGGGTATTTGGTATCTGAATCTACTCATTTACCTACATACTGAAGATTTTGCGATCGTCTAATATATTGGACTAATGCCCGATTTCTGATCAATTACTCTAGGCGATACTTCATCGCTGGCCTTATTTGGATTTTGCTCAAGTGCTAAACTCTCTGCGCGTCAATACTAGTCTGACATCAGTCAAGACCTGCTATCTGAAAACTACTAGAGAGATATACCTAACAACTTTAGTGGATAAATCAGGTCTGGAGATTGTCATATAATGCCACTAGGGTCAGAAGGCTGTGTCAAAGTTAGTGGTTAGTAGGTCTCCGCTCTGCGGTACTATTCTTATATTCTCTTACTATGCATCAAACAAAATAGAATGCATAGACAAACCGCCTGCCAAGTTTACAAGATAACTTGCGTATAGGTTTATAAGGGTTCTTCTGTATCGCTCTCACTCGCACT CTCACGCACA.

Optimal concentrations of primers and polymerase were first determinedfor the intermediate length 300-bp product using a 4 second combinedannealing/extension segment with 4.9 seconds per cycles (FIG. 10a ).Identical primer (4 μM) and polymerase (2 μM) concentrations were thenused for all product lengths and minimum extension times were determined(FIG. 11a-e ). Depending on the product length, increased extensiontimes resulted in decreased fractional quantification cycles (Cq) untilno further change was observed, reflecting the minimum extension timerequired for efficient PCR. For example, amplification curves using theKAPA2G™ FAST polymerase (Kapa Biosystems) for the 500 bp product areshown in FIG. 10b . The minimum extension time using KAPA2G FASTpolymerase was 3 s, compared to 7 s using KlenTaq1 (a deletion mutant ofTaq polymerase, AB Peptides). When the identity of the polymerase iskept constant, longer products required longer extension times (FIG. 10c). For KlenTaq1 polymerase, about 1 second is required for each 60 bps,while for KAPA2G FAST, 1 second is required for each 158 bp. It is notedthat these two polymerases were chosen because they are commerciallyavailable at sufficient concentrations, while most other polymerases arenot commercially available at such high concentrations. It is understoodthat the required time for extension depends directly and linearly withthe length to be extended, and inversely with the concentration ofpolymerase and the polymerase speed. A proportionality constant (k2) canbe defined that relates these 3 parameters:

Required Extension Time=k2*(extension length)/([polymerase]*(polymerasespeed))

Example 9

Extreme PCR times can also be reduced with high Mg⁺⁺ concentrations. A60 bp fragment of AKAP10 was amplified with primers:GCTTGGAAGATTGCTAAAATGATAGTCAGTG (SEQ ID NO:25) andTTGATCATACTGAGCCTGCTGCATAA (SEQ ID NO:26), to generate the ampliconGCTTGGAAGATTGCTAAAATGATAGTCAGTGAC(A/G)TTATGCAGCAGGCTCA GTATGATCAA (SEQID NO:27).

Each reaction was in a 1 μl volume with time based control (0.07 secondsin a 94° C. water bath, 0.1-0.4 seconds in a 60° C. water bath) for 35cycles using 2-7 mM MgCl₂. The sample volume was 1 μl, with 5 ng humangenomic DNA, 20 μM primers, and 8 μM polymerase. Using a 0.42 second percycle protocol, when the MgCl₂ was 2-3 mM, no product was observed onmelting curves (FIG. 12a ) or gels (FIG. 12b ). Minimal product waspresent at 4 mM, but a large amount of product was observed afteramplification with 5-7 mM MgCl₂. At 5 mM MgCl₂, no products wereobserved on melting curves (FIG. 13a ) or gels (FIG. 13b ) with cycletimes of 0.32 seconds, but large amounts of product were present atcycle times of 0.42 seconds, 0.52 seconds, and 0.62 seconds,demonstrating that specific, high yield 60 bp products can be obtainedin PCR performed in under 15 seconds (35 cycles in 14.7 seconds). Thus,illustrative Mg++ concentrations are at least 4 mM, at least 5 mM, atleast 6 mM, at least 7 mM, or more, and it is understood that theseillustrative Mg++ concentrations may be used with any of the embodimentsdescribed herein.

Example 10

The high concentrations of primer and polymerase used in extreme PCR canhave detrimental effects when used at slower cycling speeds.Non-specific products were obtained on rapid cycle or block basedinstruments that are 32- or 106-fold slower, respectively. FIG. 14a-bshows the results comparing amplification of the AKAP10 60 bp productused in Example 9, wherein amplification was performed using 20 μM ofeach primer, 8 μM KlenTaq and 10 ng human genomic DNA for 40 cyclesusing: (1) extreme PCR with set times of 0.5 s at 94° and 0.2 seconds at60° giving a total time of approximately 17 seconds, (2) Rapid cycle PCR(Roche LightCycler) using set times of 10 s at 94° for an initialdenaturation, followed by cycles of 85° for 0 seconds, and 60° for 0seconds, giving a total time of approximately 9 minutes, and (3) Legacy(block) temperature cycling (BioRad CFX96) with a 10 s initialdenaturation at 94°, following by temperature cycling for 0 s at 85° and5 s at 60° with a total time of approximately 30 minutes. As can beseen, even the rapid cycling of the LightCycler resulted in quite a bitof non-specific amplification, while the extreme cycling conditionsresulted in a single melting peak and minimal non-specific amplificationon the gel.

It also noted that the yield is enhanced in extreme PCR, resulting fromhigh primer and polymerase concentrations. Extreme PCR produced over30-fold the amount of product compared to rapid cycle PCR, usingquantitative PCR for comparison (data not shown).

Examples 1-10 were all performed using one or more of the devicesdescribed in FIGS. 1a-1d , or minor variations on those configurations.However, it is understood that the methods and reactions describedherein may take place in a variety of instruments. The water baths andtubes used in these examples allow for sufficiently rapid temperaturechange to study the effects of elevated concentrations of primers andpolymerase. However, other embodiments may be more suitablecommercially. Microfluidics systems, with low volume and high surfacearea to volume ratios, may be well suited to extreme PCR. Such systemsallow for rapid temperature changes required by the high concentrationsof primers and polymerase that are used in extreme PCR. Microfluidicssystems include micro-flow systems (35, 53) that incorporateminiaturized channels that repeatedly carry the samples throughdenaturation, annealing, and extension temperature zones. Some of thesesystems have already demonstrated effective PCR with cycle times as fastas 3 seconds for lower complexity targets. It is expected that morecomplex targets may be amplified in such systems if the polymerase isprovided at a concentration of at least 0.5 μM and primers are eachprovided at a concentration of at least 2 μM. Stationary PCR chips andPCR droplet systems (54) may also benefit from increased primer andprobe concentrations, as the volumes may be as small as 1 nl or smallerand may be low enough to permit very fast cycling. It is understood thatthe exact instrumentation is unimportant to the present invention,provided that the instrumentation temperature cycles fast enough to takeadvantage of increased primer and polymerase concentrations withoutsuffering from the loss of specificity associated with higher primerconcentrations at slower cycle speeds.

While the above examples all employ PCR, it is understood that PCR isillustrative only, and increased primer and enzyme concentrationscombined with shorter amplification times are envisioned for nucleicacid amplification methods other than PCR. Illustrative enzymaticactivities whose magnitude may be increased include polymerization (DNApolymerase, RNA polymerase or reverse transcriptase), ligation, helicalunwinding (helicase), or exonuclease activity (5′ to 3′ or 3′ to 5′),strand displacement and/or cleavage, endonuclease activity, and RNAdigestion of a DNA/RNA hybrid (RNAse H). Amplification reactions includewithout limitation the polymerase chain reaction, the ligase chainreaction, transcription medicated amplification (includingtranscription-based amplification system, self-sustained sequencereplication, and nucleic acid sequence-based amplification), stranddisplacement amplification, whole genome amplification, multipledisplacement amplification, antisense RNA amplification, loop-mediatedamplification, linear-linked amplification, rolling circleamplification, ramification amplification, isothermal oligonucleotideamplification, helicase chain reaction, and serial invasive signalamplification.

In general, as the enzyme activity is varied, the amplification timevaries inversely by the same factor. For reactions that include primers,as the primer concentration is varied, the amplification time variesinversely by the same factor. When both primers and enzymes are requiredfor amplification, both enzyme and primer concentrations should bevaried in order to maximize the reaction speed. If primer annealingoccurs in a unique segment of the amplification cycle (for example, aunique temperature during 3-temperature PCR), then the time required forsatisfactory completion of primer annealing in that segment is expectedto be inversely related to the primer concentration. Similarly, if theenzyme activity is required in a unique segment of the amplificationcycle (for example, a unique temperature during 3-temperature PCR), thenthe time required for satisfactory completion of the enzymatic processin that segment is expected to be inversely related to the enzymeconcentration within a certain range. Varying the primer or enzymeconcentrations can be used to change the required times of theirindividual segments, or if both occur under the same conditions (such asin 2-temperature PCR or during an isothermal reaction process), it isexpected that a change in both concentrations may be necessary toprevent one reaction from limiting the reaction speed. Increased Mg++concentration can also be used in combination with increased enzyme andprimer concentrations to further speed amplification processes. HigherMg++ concentrations both increase the speed of primer annealing andreduce the time for many enzymatic reactions used in nucleic acidamplification.

Higher concentrations of Mg++, enzymes, and primers are particularlyuseful when they are accompanied by shorter amplification times orsegments. When higher concentrations are used without shortening times,non-specific amplification products may occur in some cases, as the“stringency” of the reaction has been reduced. Reducing theamplification time or segment time(s) introduces a higher stringencythat appears to counterbalance the loss of stringency from increasedreactant concentrations. Conversely, reagent costs can be minimized byreducing the concentration of the reactants if these lowerconcentrations are counterbalanced by increased amplification times orsegment times.

Increasing polymerase concentrations can reduce the time necessary forlong-range PCR, illustratively where the target is 5-50 kb. Typically,10 min to 30 min extension periods are used to amplify large targetsbecause the target is so long that such times are needed: 1) for thepolymerase to complete extension of a single target, and 2) for enzymerecycling to polymerize additional primed templates. This recycling ofpolymerase is not needed at the beginning of PCR, when the availableenzyme outnumbers the primed template molecules. However, even beforethe exponential phase is finished, the number of polymerase moleculesoften becomes limiting and enzyme recycling is necessary. By increasingthe concentration of the polymerase, the required extension period canbe reduced to less than 5 minutes and possibly less than 2 minutes,while maintaining increased yield due to the high primer concentration.Although the actual enzyme speed is not increased, less recycling isnecessary, affecting the minimum time required, approximately in alinear fashion with the enzyme concentration.

Cycle sequencing times can also be reduced by increasing primer andpolymerase concentrations. Typically, in standard cycle sequencingprimer concentrations are 0.16 μM and the combined annealing/extensionperiod is 10 min at 50-60 degrees C. By increasing the primer andpolymerase concentrations by 10-fold, the time required forannealing/extension can be reduced approximately 10-fold. In both longPCR and cycle sequencing, the expected time required is inverselyproportional to the polymerase or primer concentration, whichever islimiting.

PCR of fragments with ligated linkers that are used as primers inpreparation for massively parallel sequencing can be completed in muchless time than currently performed by combining extreme temperaturecycling with higher concentrations of primers, polymerase, and/or Mg++.

In all of the above applications, it is expected that the specificity ofthe reaction is maintained by shorter amplification times. Although highprimer and polymerase concentrations are expected by those well versedin the art to cause difficulty from non-specific amplification,minimizing the overall cycle time and/or individual segment timesresults in high specificity and efficiency of the PCR.

TABLE 2 Target KCNE1 KCNE1 IRL10RB IRL10RB IRL10RB NQO1 AKAP10 SyntheticAmplicon Size (bp) 45 45 49 49 58 102 60 100 Polymerase KlenTaq 1KlenTaq1 KlenTaq1 KlenTaq1 KlenTaq1 KlenTaq1 KlenTaq1 KlenTaq1[Polymerase] 1 8 4 8 2 2 8 2 [Primers] 10 20 10 20 10 8 20 4 # Cycles 35RT 35 35 39 30 35 RT Cycle Time (s) 0.8 0.91 0.73 0.45 0.97 1.93 0.421.9 PCR Time (s) 28 RT 26 16 38 58 14.7 RT Hot Water Temp 95.5 95.5 95.595.5 95.5 95.5 95.5 95.5 (° C.) Cold Water Temp 20 58 30 30 30 72 59 76(° C.) Hot Trigger Temp 90 85 90 90 90 90 Time 92 (° C.) Cold TriggerTemp 70 62 70 70 70 Time Time Time (° C.) Denaturation (° C.) 90 85 9090 90 90 (82-85) 92 w/ TC Ann/Ext (° C.) 60 60 65 65 65 72 60 76 Ann/ExtTime (s) 0 0 0 0 0 1 0.1-0.4 0.5-3 Figure 9a 9a 5a 5a 4c 7a 12a, 11a Tm81 81 80 80 83 85 79 85 Mg++ 3 3 3 3 3 3 2-7 3 Target SyntheticSynthetic Synthetic Synthetic Synthetic Synthetic Amplicon Size (bp) 200300 300 400 500 500 Polymerase KlenTaq1 KlenTaq1 KlenTaq1 KlenTaq1KlenTaq1 KAPA2G FAST [Polymerase] 2 2 2 2 2 2 [Primers] 4 4 4 4 4 4 #Cycles RT 20 RT RT RT RT Cycle Time (s) 3.9 4.9 5.9 7.9 7.9 3.9 PCR Time(s) RT 98 RT RT RT RT Hot Water Temp 95.5 95.5 95.5 95.5 95.5 95.5 (°C.) Cold Water Temp 76 76 76 76 76 76 (° C.) Hot Trigger Temp 92 92 9292 92 92 (° C.) Cold Trigger Temp Time Time Time Time Time Time (° C.)Denaturation (° C.) 92 92 92 92 92 92 Ann/Ext (° C.) 76 76 76 76 76 76Ann/Ext Time (s) 1-5 4 1-7 3-9 3-11 1-5 Figure 11b 10a, 11c 11c 11d 11e10b Tm 85 85 85 81/87 (2 84 84 domains) Mg++ 3 3 3 3 3 3 Time =time-based segment control does not have a temperature trigger RT =real-time acquisition

TABLE 3 Extension Anneal/ [Primer] [Polymerase] Polymerase Length CycleExtend (μM) (μM) Speed (nt/s) (bp) Time (s) Time (s) [Mg++] Standard0.05-0.5  0.0026-0.026  10-45 20-980 120-480 15-60 1.5 Rapid Cycle0.2-1.0 0.063 55-90 20-480 20-60  1-10 3 Extreme  1-16 0.5-8    50-10020-280 0.5-5   <0.1-5     3-7 Opt Extreme #1 10 2.50 60 29 0.73 <0.1 3Opt Extreme #2 4 0.50 60 82 1.93 1 3 Opt Extreme #3 4 0.75 60 280 4.9 43 If Required Annealing time = k1/[primer] [Primer] Anneal/Extend k1 k1range (μM) Time (s) (s * μM) (s * μM) Min Standard 0.05 15 0.75 Standard0.75-30 Max Standard 0.5 60 30 Rapid Cycle  0.2-10 Min Rapid Cycle 0.2 10.2 Extreme   1-20 Max Rapid Cycle 1 10 10 Opt Extreme #1 10 0.1 1 OptExtreme #2 4 1 4 Opt Extreme #3 4 5 20 If required extension time = k2 *product length/(polymerase speed * [polymerase]) Extension Anneal/[Polymerase] Polymerase Length Extend k2 (μM) Speed (nt/s) (bp) Time (s)(1/μM) Opt Extreme #1 2.5 60 29 0.1 0.52 Opt Extreme #2 0.5 60 82 1 0.37Opt Extreme #3 0.75 60 280 4 0.64

Specific conditions for extreme PCR are shown in Table 2. All data arepresented except for the simultaneous optimization experiments forpolymerase and primer concentrations for 3 of the targets. In Table 3,the quantitative relationships between variables are detailed. Theinverse proportionality that relates the required annealing time to theprimer concentration is approximately constant (k1) and defined by theequation (Required annealing time)=k1/[primer]. Using a range of typicalvalues for these variables under conditions of legacy (standard) PCR,rapid cycle PCR, and extreme PCR produces ranges for the inverseproportionality constant that largely overlap (legacy 0.75-30, rapidcycle 0.2-10, and extreme 1-20). Because of this constant inverseproportionality, desired annealing times outside of those currentlyperformed can be used to predict the required primer concentrations forthe desired time. For example, using a constant of 5 (s*μM), for anannealing time of 0.01 s, a primer concentration of 500 μM can becalculated. Conversely, if a primer concentration of 0.01 μM weredesired, the required annealing time would be 500 seconds. Althoughthese conditions are outside the bounds of both legacy and extreme PCR,they predict a relationship between primer concentrations and annealingtimes that are useful for PCR success. Reasonable bounds for k1 acrosslegacy, rapid cycle and extreme PCR are 0.5-20 (s×μM), more preferred1-10 (s×μM) and most preferred 3-6 (s×μM).

Similar calculations can be performed to relate desired extension timesto polymerase concentration, polymerase speed, and the length of theproduct to be amplified. However, because of many additional variablesthat affect PCR between legacy, rapid cycle and extreme PCR (polymerase,Mg++, buffers), performed in different laboratories over time, it may bebest to look at the well-controlled conditions of extreme PCR presentedhere to establish an inverse proportionality between variables. Thisallows a quantitative expression between polymerase concentration,polymerase speed, product length, and the required extension time underextreme PCR conditions. The defining equation is (Required ExtensionTime)=k2(product length)/([polymerase]*(polymerase speed)). Theexperimentally determined k2 is defined as the proportionality constantin the above equation under conditions of constant temperature, Mg⁺⁺,type of polymerase, buffers, additives, and concentration of dsDNA dye.For the 3 extreme PCR targets with two dimensional optimization of[polymerase] and [primer], the [polymerase] at the edge of successfulamplification can be discerned across primer concentrations and relatedto the other 3 variables. As shown in Table 3, the values of k2 forthese 3 different targets vary by less than a factor of 2, from which itis inferred that k2 is a constant and can be used to predict onevariable if the others are known. The required extension time isproportional to the extension length (product length minus the primerlength) and inversely proportional to the polymerase speed andconcentration of polymerase. k2 has units of (1/μM) and an optimal valuefor the extreme PCR conditions used here of 0.5 (1/μM) with a range of0.3-0.7 (1/μM). Similar values for k2 could be derived for otherreaction conditions that vary in polymerase type, Mg++ concentration ordifferent buffer or dye conditions.

Extreme PCR can be performed in any kind of container, as long as thetemperature of the sample can be changed quickly. In addition tostandard tubes and capillaries, micro-droplets of aqueous reactionssuspended in an oil stream or thin 2-dimensional wafers provide goodthermal contact. Continuous flow PCR of a sample stream (eitherdispersed as droplets, separated by bubbles, or other means to preventmixing) past spatial segments at different temperatures is a good methodfor the temperature control needed for the speeds of extreme PCR.

REFERENCES

-   1. Wittwer C T, Reed G B, Ririe K M. Rapid cycle DNA amplification.    In: Mullis I K, Ferre F, Gibbs R, eds. The polymerase chain    reaction, Vol. Deerfield Beach, F L: 174-181, 1994.-   2. Wittwer C T, Fillmore G C, Hillyard D R. Automated polymerase    chain reaction in capillary tubes with hot air. Nucleic Acids Res    1989; 17:4353-7.-   3. Wittwer C T, Fillmore G C, Garling D J. Minimizing the time    required for DNA amplification by efficient heat transfer to small    samples. Anal Biochem 1990; 186:328-31.-   4. Wittwer C T, Garling D J. Rapid cycle DNA amplification: time and    temperature optimization. Biotechniques 1991; 10:76-83.-   5. Wittwer C T, Marshall B C, Reed G H, Cherry J L. Rapid cycle    allele-specific amplification: studies with the cystic fibrosis    delta F508 locus. Clin Chem 1993; 39:804-9.-   6. Schoder D, Schmalwieser A, Schauberger G, Hoorfar J, Kuhn M,    Wagner M. Novel approach for assessing performance of PCR cyclers    used for diagnostic testing. J Clin Microbiol 2005; 43:2724-8.-   7. Herrmann M G, Durtschi J D, Wittwer C T, Voelkerding K V.    Expanded instrument comparison of amplicon DNA melting analysis for    mutation scanning and genotyping. Clin Chem 2007; 53:1544-8.-   8. Herrmann M G, Durtschi J D, Bromley L K, Wittwer C T, Voelkerding    K V. Amplicon DNA melting analysis for mutation scanning and    genotyping: cross-platform comparison of instruments and dyes. Clin    Chem 2006; 52:494-503.-   9. Raja S, El-Hefnawy T, Kelly L A, Chestney M L, Luketich J D,    Godfrey T E. Temperature-controlled primer limit for multiplexing of    rapid, quantitative reverse transcription-PCR assays: application to    intraoperative cancer diagnostics. Clin Chem 2002; 48:1329-37.-   10. Wittwer C T, Ririe K M, Andrew R V, David D A, Gundry R A, Balis    U J. The LightCycler: a microvolume multisample fluorimeter with    rapid temperature control. Biotechniques 1997; 22:176-81.-   11. Wittwer C T, Ririe K M, Rasmussen R P. Fluorescence monitoring    of rapid cycle PCR for quantification. In: Ferre F, ed. Gene    Quantification, New York: Birkhauser, 1998:129-44.-   12. Elenitoba-Johnson O, David D, Crews N, Wittwer C T. Plastic vs    glass capillaries for rapid-cycle PCR. Biotechniques 2008;    44:487-8,490,492.-   13. Roper M G, Easley C J, Landers J P. Advances in polymerase chain    reaction on microfluidic chips. Anal Chem 2005; 77:3887-93.-   14. Zhang C, Xing D. Miniaturized PCR chips for nucleic acid    amplification and analysis: latest advances and future trends.    Nucleic Acids Res 2007; 35:4223-37.-   15. Cheng J, Shoffner M A, Hvichia G E, Kricka L J, Wilding P. Chip    PCR. I I. Investigation of different PCR amplification systems in    microfabricated silicon-glass chips. Nucleic Acids Res 1996;    24:380-5.-   16. Woolley A T, Hadley D, Landre P, deMello A J, Mathies R A,    Northrup M A. Functional integration of PCR amplification and    capillary electrophoresis in a microfabricated DNA analysis device.    Anal Chem 1996; 68:4081-6.-   17. Neuzil P, Zhang C, Pipper J, Oh S, Zhuo L. Ultra fast    miniaturized real-time PCR: 40 cycles in less than six minutes.    Nucleic Acids Res 2006; 34:e77.-   18. Oda R P, Strausbauch M A, Huhmer A F, Borson N, Jurrens S R,    Craighead J, et al. Infrared-mediated thermocycling for ultrafast    polymerase chain reaction amplification of DNA. Anal Chem 1998;    70:4361-8.-   19. Roper M G, Easley C J, Legendre L A, Humphrey J A, Landers J P.    Infrared temperature control system for a completely noncontact    polymerase chain reaction in microfluidic chips. Anal Chem 2007;    79:1294-300.-   20. Friedman N A, Meldrum D R. Capillary tube resistive thermal    cycling. Anal Chem 1998; 70:2997-3002.-   21. Heap D M, Herrmann M G, Wittwer C T. PCR amplification using    electrolytic resistance for heating and temperature monitoring.    Biotechniques 2000; 29:1006-12.-   22. Kopp M U, Mello A J, Manz A. Chemical amplification:    continuous-flow PCR on a chip. Science 1998; 280:1046-8.-   23. Hashimoto M, Chen P C, Mitchell M W, Nikitopoulos D E, Soper S    A, Murphy M C. Rapid PCR in a continuous flow device. Lab Chip 2004;    4:638-45.-   24. Crews N, Wittwer C, Gale B. Continuous-flow thermal gradient    PCR. Biomed Microdevices 2008; 10; 187-95.-   25. Chiou J T, Matsudaira P T, Ehrlich D J. Thirty-cycle temperature    optimization of a closed-cycle capillary PCR machine. Biotechniques    2002; 33:557-8, 60, 62.-   26. Frey O, Bonneick S, Hierlemann A, Lichtenberg J. Autonomous    microfluidic multi-channel chip for real-time PCR with integrated    liquid handling. Biomed Microdevices 2007; 9:711-8.-   27. Chen J, Wabuyele M, Chen H, Patterson D, Hupert M, Shadpour H,    et al. Electrokinetically synchronized polymerase chain reaction    microchip fabricated in polycarbonate. Anal Chem 2005; 77:658-66.-   28. Sun Y, Kwok Y C, Nguyen N T. A circular ferrofluid driven    microchip for rapid polymerase chain reaction. Lab Chip 2007;    7:1012-7.-   29. Agrawal N, Hassan Y A, Ugaz V M. A pocket-sized convective PCR    thermocycler. Angew Chem Int Ed Engl 2007; 46:4316-9.-   30. Zhang C, Xu J, Ma W, Zheng W. PCR microfluidic devices for DNA    amplification. Biotechnol Adv 2006; 24:243-84.-   31. Wheeler E K, Benett W, Stratton P, Richards J, Chen A, Christian    A, et al. Convectively driven polymerase chain reaction thermal    cycler. Anal Chem 2004; 76:4011-6.-   32. Belgrader P, Benett W, Hadley D, Long G, Mariella R, Jr.,    Milanovich F, et al. Rapid pathogen detection using a microchip PCR    array instrument. Clin Chem 1998; 44:2191-4.-   33. Terazona H, Takei, H, Hattori A, Yasuda K. Development of a    high-speed real-time polymerase chain reaction system using a    circulating water-based rapid heat exchange. Jap J Appl Phys 2010;    49:06GM05.-   34. Wheeler E K, Hara C A, Frank J, Deotte J, Hall S B, Benett W,    Spadaccini C, Beer N R. Under-three minute PCR: Probing the limits    of fast amplification. Analyst 2011.-   35. Fuchiwaki Y, Nagai H, Saito M, Tamiya E. Ultra-rapid    flow-through polymerase chain reaction microfluidics using vapor    pressure. Biosens Bioelect 2011; 27:88-94.-   36. Maltezos G, Johnston M, Taganov K, Srichantaratsamee C, Gorman    J, Baltimore D, Chantratita W and Scherer A, Appl. Phys. Lett.,    2010, 97, 264101.-   37. Wilhelm J, Hahn M, Pingoud A. Influence of DNA target melting    behavior on real-time PCR quantification. Clin Chem 2000;    46:1738-43.-   38. Zuna J, Muzikova K, Madzo J, Krejci 0, Trka J. Temperature    non-homogeneity in rapid airflow-based cycler significantly affects    real-time PCR. Biotechniques 2002; 33:508, 10, 12.-   39. von Kanel T, Adolf F, Schneider M, Sanz J, Gallati S. Sample    number and denaturation time are crucial for the accuracy of    capillary-based LightCyclers. Clin Chem 2007; 53:1392-4.-   40. Wittwer C T, Herrmann M G. Rapid thermal cycling and PCR    kinetics. In: Innis M, Gelfand D, Sninsky J, eds. PCR Methods    Manual, Vol. San Diego: Academic Press, 1999:211-29.-   41. Wittwer C T, Reed G H, Gundry C N, Vandersteen J G, Pryor R J.    High-resolution genotyping by amplicon melting analysis using    LCGreen. Clin Chem 2003; 49:853-60.-   42. von Ahsen N, Wittwer C T, Schutz E. Oligonucleotide melting    temperatures under PCR conditions: nearest-neighbor corrections for    Mg(2+), deoxynucleotide triphosphate, and dimethyl sulfoxide    concentrations with comparison to alternative empirical formulas.    Clin Chem 2001; 47:1956-61.-   43. Ririe K M, Rasmussen R P, Wittwer C T. Product differentiation    by analysis of DNA melting curves during the polymerase chain    reaction. Anal Biochem 1997; 245:154-60.-   44. Wittwer C T, Herrmann M G, Moss A A, Rasmussen R P. Continuous    fluorescence monitoring of rapid cycle DNA amplification.    Biotechniques 1997; 22:130-1, 4-8.-   45. Weis JI-1, Tan S S, Martin B K, Wittwer C T. Detection of rare    mRNAs via quantitative R T-PCR. Trends Genet 1992; 8:263-4.-   46. Brown R A, Lay M J, Wittwer C T. Rapid cycle amplification for    construction of competitive templates. In: Horton R M, Tait R C,    eds. Genetic Engineering with PCR, Vol. Norfolk: Horizon Scientific    Press, 1998:57-70.-   47. Ausubel et al., Current Protocols in Molecular Biology, John    Wiley & Sons, New York, N.Y., 1998-   48. Whitney S E, “Analysis of rapid thermocycling for the polymerase    chain reaction,” Ph.D. thesis, University of Nebraska, 2004.-   49. Lawyer F C, Stoffel S, Saiki R K, Chang S Y, Landre P A,    Abramson R D, Gelfand D H. High-level expression, purification and    enzymatic characterization of full-length Thermus aquaticus DNA    polymerase and a truncated form deficient of 5′ to 3′ exonuclease    activity. PCR Meth Appl. 1993; 2:275-287.-   50. Innis M A, Myamo K B, Gelfand D H, Brow MAD. DNA sequencing with    Thermus aquaticus DNA polymerase and direct sequencing of polymerase    chain reaction-amplified DNA. Proc. Natl. ACad. Sci USA 1988;    85:9436-40.-   51. Terazono H, Hattori A, Takei H, Takeda K, Yasuda K. Development    of 1480 nm photothermal high-speed real-time polymerase chain    reaction system for rapid nucleotide recognition. Jpn J Appl Phys.    2008; 47:5212-6.-   52. Wittwer C T, Rasmussen R P, Ririe K M. Rapid PCR and melting    curve analysis. In: The PCR Revolution: Basic Technologies and    Applications, Bustin S A, ed. Cambridge Univ Press, New York, 48-69,    2010.-   53. Fuchiwaki Y, Saito M, Wakida S, Tamiya E, Nagai H. A practical    liquid plug flow-through polymerase chain-reaction system based on a    heat-resistant resin chip. Anal Sci. 2011; 27:225-30.-   54. Kim H, Dixit S, Green C J, Faris G W. Nanodroplet real-time PCR    system with laser assisted heating. Optics Express 2009; 17:218-27.-   55. Obeid P J, Christopoulos T K, Crabtree H J, Backhouse C J.    Microfabricated device for DNA and RNA amplification by    continuous-flow polymerase chain reaction and reverse    transcription-polymerase chain reaction with cycle number selection.    Anal Chem 2003; 75:288-95.-   56. Giordano B C, Ferrance J, Swedberg S, Huhmer A F R, Landers J P.    Polymerase chain reaction in polymeric microchips: DNA amplification    in less than 240 seconds. Anal Biochem 2001; 291:124-132.

Although the invention has been described in detail with reference topreferred embodiments, variations and modifications exist within thescope and spirit of the invention as described and defined in thefollowing claims.

That which is claimed is:
 1. A kit for performing PCR on a targetnucleic acid, the kit comprising: dNTPs, a polymerase provided at aconcentration of at least 0.5 μM, and a pair of primers configured foramplifying the target nucleic acid, wherein each primer of the pair ofprimers are provided at a concentration of at least 2 μM.
 2. The kit ofclaim 1, wherein the concentration for each primer of the pair ofprimers is greater than 2.5 μM.
 3. The kit of claim 1, furthercomprising a Mg⁺⁺ concentration of at least 4 mM.
 4. The kit of claim 1,further comprising instructions for performing PCR using a temperaturecycling profile wherein each cycle is completed in a cycle time lessthan 20 seconds per cycle.
 5. The kit of claim 4, wherein the cycle timeis less than 10 seconds per cycle.
 6. The kit of claim 1, wherein thepolymerase is KlenTaq provided at the concentration of at least 1.0 μM.7. The kit of claim 1, wherein the target nucleic acid is eukaryotic andthe kit is configured for amplifying from eukaryotic genomic DNA.
 8. Akit for performing PCR on a target nucleic acid, the kit comprising:dNTPs, a thermostable polymerase, and primers configured foramplification of the target nucleic acid sequence to the biologicalsample, wherein the polymerase to primer ratio in the kit is (0.03 to0.4 polymerase):(total primer concentration) and the polymerase has aconcentration of at least 0.5 μM.
 9. The kit of claim 8, wherein theprimers have a total concentration of at least 5 μM.
 10. The kit ofclaim 8, wherein the polymerase is KlenTaq provided at the concentrationof at least 1.0 μM.
 11. The kit of claim 8, further comprisinginstructions for performing PCR using a temperature cycling profilewherein each cycle is completed in a cycle time less than 20 second percycle.
 12. The kit of claim 11, wherein the cycle time is less than 10seconds per cycle.
 13. The kit of claim 8, wherein the target nucleicacid is eukaryotic and the kit is configured for amplifying fromeukaryotic genomic DNA.